TECHNICAL FIELD
[0001] The present invention relates to a method of isolating a nucleic acid having an intended
functional property and a kit for carrying out the method.
BACKGROUND ART
[0002] Expression cloning is a method of cloning a gene that uses a function exhibited as
a result of expression of the target gene as an index. This method does not requires
information such as the base sequence of the gene or the amino acid sequence of the
gene product, and is advantageous when cloning genes whose expression amount is small,
and/or genes for which only functional information is available.
[0003] The gene products of mammals include many proteins that interact with other factors
(such as proteins) in mammalian cells. Therefore, when conducting expression cloning
of genes from mammalian cells, it is preferable to use the mammalian cells from which
the gene is derived.
[0004] In the case where expression cloning is conducted using a mammalian cell according
to conventional methods, even when a mammalian cell containing nucleic acid having
the intended functional property is isolated, usually the isolated mammalian cell
simultaneously contains plural kinds of nucleic acids in a single cell, in contrast
to prokaryotes and fungi. For this reason, it is necessary to purify the nucleic acid
having an intended functional property from the plurality of nucleic acids existing
in the isolated cell.
[0005] When conventional methods are used, a large amount of labor is required in order
to purify a nucleic acid having an intended functional property from an isolated cell.
This may be attributed to the following fact; during expression screening using mammalian
cells, a known retrovirus or adenovirus is used as a vector system to stably carry
a foreign gene, however, these virus vector systems incorporate several vector genes
into the chromosome of a host cell, thus in order to purify and identify the transferred
nucleic acid the purification process has to be repeated several times. In order to
obtain a purified clone, a typical purification process comprises the steps of:
1) PCR amplification of the nucleic acid sequence;
2) transfection of the host cell with the amplified PCR fragment; and
3) selection of transfected cells based on a desired phenotype, and the series of
the steps should be repeated 10 to 20 times
[0006] Methods of cloning and purifying a mixture of nucleic acids using microorganisms
such as Escherichia coli are well known. Theoretically, after isolating a first host
cell containing plural kinds of nucleic acids, a specific nucleic acid may be purified
from the plural kinds of transferred nucleic acids using a second host cell such as
Escherichia coli. Such purification methods require, for example, the steps of amplifying
a nucleic acid incorporated into a chromosome by PCR or the like; ligating the amplified
nucleic acid into a vector that autonomously replicates in a second host cell; and
transferring the ligated product into a second host cell. However, this ligation step
is very inefficient, and as the number of individual nucleic acids to be ligated increases,
the chances of succeeding in purifying the target nucleic acid reduces. For this reason,
in conventional methods using retroviruses, adenoviruses and the like, a second host
cell such as Escherichia coli is not used for the purpose of purifying a specific
nucleic acid, for example, during expression cloning.
[0007] As a means for transferring a foreign nucleic acid into a mammalian cell, besides
the method of using viruses such as retroviruses, conventional methods such as calcium
precipitation are also well-known. However, when transient expression is conducted
using conventional methods such as calcium precipitation, unlike methods using retroviruses,
adenoviruses or the like, degradation of the nucleic acid occurs during transduction,
and the nucleic acid fragment encoding the transferred foreign gene is unstable in
the host cell. These problems are due to an intrinsic complication of conventionally
used methodsof transient expression, and thus transient expression systems such as
calcium precipitation are thought to be unsuited as means for obtaining a clone.
[0008] Eventually, in conventional methods, even if a candidate nucleic acid is isolated,
the candidate gene is usually a population of clones including a plurality of different
clones, thus it is necessary to purify and isolate a single clone from the population
of clones. This purification requires a large amount of labor in terms of multiple
screening steps, and this obstructs practical isolation and screening.
[0009] Therefore, it is an object of the present invention to provide a novel method of
simply and rapidly isolating a nucleic acid (e.g., screening method), and to provide
a kit for carrying out the method.
SUMMARY OF THE INVENTION
[0010] Therefore, the present invention provides a novel method of isolating a nucleic acid
conveniently and rapidly (e.g., screening method), as well as a kit for carrying out
the method. Byusing the method and kit of the present invention, it is possible to
carry out screening, particularly expression screening using mammalian cells, rapidly
and conveniently. Conventionally, during expression screening usingmammalian cells,
a large amount of labor and time was required in order to purify a candidate nucleic
acid, however the required labor and time can largely be reduced using the present
invention. Therefore, the effect of the present invention is significant.
[0011] The present invention has the following features:
(1) A method of isolating a nucleic acid having an intended functional property, comprising
the steps of:
(A) transferring a nucleic acid into a plurality of first host cells and allowing
the nucleic acid to transiently express therein;
(B) selecting, from the plurality of first host cells into which the nucleic acid
is transferred, a cell into which a nucleic acid having an intended functional property
has been transferred;
(C) preparing a purified nucleic acid from the selected cell; and
(D) selecting a purified nucleic acid having an intended functional property.
(2) The method according to item (1), wherein at least two kinds of nucleic acids
are transferred into the plurality of first host cells.
(3) The method according to item (1), wherein the step of transferring a nucleic acid
into the plurality of first host cells is carried out according to a procedure selected
from the group consisting of: a transferring method using a viral envelope, a transferring
method using a liposome, a transferring method using a liposome containing at least
one protein from a viral envelope, a transferring method using calcium phosphate and
an electroporation method.
(4) The method according to item (1), wherein the nucleic acid includes a foreign
gene and a promoter.
(5) The method according to item (1), wherein the host cells are mammalian cells.
(6) The method according to item (1), wherein the host cells are human cells.
(7) The method according to item (1), wherein the viral envelope is derived from wild-type
or recombinant viruses.
(8) The method according to item (1), wherein the viral envelope is derived from a
virus belonging to a family selected from the group consisting of Retroviridae, Togaviridae,
Coronaviridae, Flaviviridae, Paramyxoviridae, Orthomyxoviridae, Bunyaviridae, Rhabdoviridae,
Poxviridae, Herpesviridae, Baculoviridae and Hepadnaviridae.
(9) The method according to item (8), wherein the virus is derived from viruses belonging
to the family Paramyxoviridae.
(10) The method according to item (9), wherein the virus is HVJ.
(11) The method according to item (1), wherein the vector is a viral envelope vector.
(12) The method according to item (1), wherein the vector is a vector containing a
protein prepared from a viral envelope and a liposome.
(13) The method according to item (12), wherein the protein prepared from a viral
envelope is a protein selected from the group consisting of F protein, HN protein,
NP protein and a combination thereof.
(14) The method according to item (1), wherein the step (C) of preparing a purified
nucleic acid from the selected cell is carried out in the following steps of:
(i) extracting a nucleic acid from the selected cell;
(ii) transferring the extracted nucleic acid into a second host cell to thereby obtain
a transformed cell;
(iii) purifying the transformed cell; and
(iv) preparing a nucleic acid from the purified transformed cell.
(15) The method according to item (14), wherein the second host cell is a bacterium
or a fungus.
(16) The method according to item (15), wherein the nucleic acid contains a sequence
that is necessary for autonomous replication in the bacterium or fungus.
(17) The method according to item (15), wherein the bacterium belongs to a genus selected
from the group consisting of Escherichia, Bacillus, Streptococcus, Staphylococcus,
Haemophilus, Neisseria, Actinobacillus and Acinetobacter.
(18) The method according to item (17), wherein the bacterium is Escherichia coli.
(19) The method according to item (15), wherein the fungus is Saccharomyces, Schizosaccharomyces
or Neurospora.
(20) The method according to item (1), wherein the step (D) of selecting a purified
nucleic acid having an intended functional property is carried out in the following
steps of:
(i) transferring the purified nucleic acid into a third host cell to obtain a transformed
cell;
(ii) comparing the property of the transformed cell with the property of a third host
cell that is not transformed; and
(iii) determining whether or not the transformed cell has an intended functional property,
as a result of the comparison.
(21) The method according to item (20), wherein the step (D) of selecting a purified
nucleic acid having an intended functional property further includes the step of (iv)
preparing a nucleic acid having an intended functional property from the selected
cell.
(22) The method according to item (20), wherein the third host cell is a mammalian
cell.
(23) The method according to item (20), wherein the third host cell is a human cell.
(24) The method according to item (20), wherein the third host cell is derived from
the same species as the species from which the first host cell is derived.
(25) The method according to item (1), wherein the intended functional property is
selected from the group consisting of induction of angiogenesis, tumor suppression,
enhancement of osteogenesis, induction of apoptosis, cytokine secretion, induction
of dendrites, suppression of arteriosclerosis, suppression of diabetes; suppression
of autoimmune diseases; suppression of Alzheimer's disease, suppression of Parkinson's
disease, protection of nerve cells and combinations thereof.
(26) A kit for isolating a nucleic acid having an intended functional property, comprising:
(A) a nucleic acid transfer vector to be transferred into a plurality of the first
host cells in order to transform said first host cells; and
(B) a second host cell for preparing a purified nucleic acid from a cell selected
from the transformed first host cells.
(27) The kit according to item (26), wherein the nucleic acid transfer vector is a
viral envelope, liposome or liposome containing at least one protein from viral envelope.
(28) The kit according to item (26), wherein the first host cells are mammalian cells.
(29) The kit according to item (26), wherein the first host cells are human cells.
(30) The kit according to item (26), wherein the viral envelope is derived from wild-type
or recombinant viruses.
(31) The kit according to item (26), wherein the viral envelope is derived from a
virus belonging to a family selected from the group consisting of Retroviridae, Togaviridae,
Coronaviridae, Flaviviridae, Paramyxoviridae, Orthomyxoviridae, Bunyaviridae, Rhabdoviridae,
Poxviridae, Herpesviridae, Baculoviridae and Hepadnaviridae.
(32) The kit according to item (26), wherein the virus is derived from viruses belonging
to the family Paramyxoviridae.
(33) The kit according to item (26), wherein the virus is HVJ.
(34) The kit according to item (26), wherein the vector is a viral envelope vector.
(35) The kit according to item (26), wherein the vector is a vector containing a protein
prepared from a viral envelope and a liposome.
(36) The kit according to item (35), wherein the protein prepared from a viral envelope
is a protein selected from the group consisting of F protein, HN protein, NP protein
and a combination thereof.
(37) The kit according to item (26), wherein the second host cell is a bacterium or
fungus.
(38) The kit according to item (26), further comprising a nucleic acid for preparing
a nucleic acid to be transferred into the first host cells.
(39) The kit according to item (26), further comprising a reagent to be used for determining
whether or not the purified nucleic acid has an intended functional property.
(40) The kit according to item (37), wherein the bacterium belongs to a genus selected
from the group consisting of Escherichia, Bacillus, Streptococcus, Staphylococcus,
Haemophilus, Neisseria, Actinobacillus and Acinetobacter.
(41) The kit according to item (40), wherein the bacterium is Escherichia coli.
(42) The kit according to item (37), wherein the fungus is Saccharomyces, Schizosaccharomyces
or Neurospora.
(43) A nucleic acid isolated by the method according to item (1).
(44) Use of a viral envelope for isolating a nucleic acid having an intended functional
property.
(45) Use of a liposome containing at least one protein from a viral envelope, for
isolating a nucleic acid having an intended functional property.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012]
Fig. 1 is a schematic showing the steps in genome screening using HVJ-E.
Fig. 2 shows the result of an HAEC cell growth assay after transferring a genomic
library gene.
Fig. 3 is a computer-generated graph, showing the cell growth states of each well
in a human heart pcDNA library screening.
Fig. 4 is a schematic showing confirmation of an insert by digesting cloned genes
using restriction enzymes.
Fig. 5 shows the result of a second cell growth assay.
Fig. 6 shows micrographs of each well at 40-fold magnification.
Fig. 7 is a graph comparing areas of cells exhibiting angiogenesis (left) and a graph
comparing the length of cells exhibiting angiogenesis (right) obtained by using an
angiogenesis quantification software.
Fig. 8 is a graph comparing joint numbers of the junctions of cells exhibiting angiogenesis
(left) and a graph comparing the numbers of paths of cells exhibiting angiogenesis
(right).
Fig. 9 is a graph comparing the effect of the clone on c-fos gene promoter activity.
DETAILED DESCRIPTION OF THE INVENTION
[0013] Throughout the following specification explaining the present invention, it is to
be understood that articles for singular forms (e.g., "a", "an", "the" in English,
"ein", "der", "das", "die" and their declined forms in German, "un", "une", "le",
"la" in French, and "un", "una", "el", "la" in Spanish, and corresponding articles
and adjectives in other languages) also imply concepts of plural forms, unless otherwise
indicated. In addition, the terms used in this specification should be understood
as being used in the sense that is generally used in the art, unless otherwise indicated.
(DEFINITION)
[0014] The term "cell" as used herein is defined in a similar manner to the broadest meaning
used in the art, and refers to a living organism which is a sub-unit of tissue of
a multicellular organism, encapsulated by a membrane structure that separates it from
the external environment, is self-reproducing and carries genetic information and
an expression system therefore.
[0015] The terms "protein", "polypeptide" and "peptide", as used herein may be interchangeably
used, and each term refers to a macromolecule (polymer) comprising a sequence of amino
acids. The term "amino acid" refers to an organic molecule having a carboxylic group
and an amino group at a carbon atom. Preferably, the amino acids used in this specification
are usually, but are not limited to, the 20 naturally occurring amino acids.
[0016] The terms "nucleic acid", "nucleic acid molecule", "polynucleotide" and "oligonucleotide"
as used herein are interchangeably used unless otherwise indicated, and each term
refers to a macromolecule (polymer) comprising a sequence of nucleotides. The term
"nucleotide" refers to a nucleoside in which the 5' moiety of ribose is a phosphate
ester. Nucleotides having a pyrimidine base or purine base (pyrimidine nucleotide
and purine nucleotide) as a base moiety are known. A polynucleotide includes deoxyribonucleic
acid (DNA) or ribonucleic acid (RNA).
[0017] This term also includes "derivative oligonucleotide" or "derivative polynucleotide".
The term "derivative oligonucleotide" or "derivative polynucleotide" is interchangeably
used, and refers to an oligonucleotide or polynucleotide which contains a derivative
of a nucleotide or an oligonucleotide in which the bond between two or more nucleotides
is not the normal one.
[0018] Concrete examples of such oligonucleotides include: 2'-O-methyl-ribonucleotide; a
derivative oligonucleotide in which a phosphodiester bond in the oligonucleotide is
changed toaphosphorothioatebond; a derivative oligonucleotide in which a phosphodiester
bond in the oligonucleotide is changed to a N3'-P5' phosphoroamidate bond; a derivative
oligonucleotide in which a ribose and phosphodiester bond in the oligonucleotide is
changed into a peptide-nucleic acid bond; a derivative oligonucleotide in which a
uracil in the oligonucleotide is substituted by C-5 propynyluracil; a derivative oligonucleotide
in which a uracil in the oligonucleotide is substituted by C-5 thiazole uracil; a
derivative oligonucleotide inwhich a cytosine in the oligonucleotide is substituted
by C-5 propynyl cytosine; a derivative oligonucleotide in which a cytosine in the
oligonucleotide is substituted by phenoxazine-modified cytosine; a derivative oligonucleotide
in which a ribose moiety in a DNA molecule is substituted by 2'-O- propyl ribose;
and a derivative oligonucleotide in which a ribose moiety in the oligonucleotide is
substituted by 2'-methoxyethoxy ribose. Unless otherwise specified, a specific nucleic
acid sequence is intended to encompass conservative variants (for example, degenerate
codon substitutes) or complementary sequences thereof as well as the specific sequence
given. Specifically, a degenerate codon substitute can be achieved by creating a sequence
in which the third position of one or more selected (or all) codon(s) is substituted
by a mixed base and/or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081(1991);
Otsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell.
Probes 8: 91-98 (1994)). The term "nucleic acid" as used herein is also used interchangeably
with gene, cDNA, RNA, mRNA, oligonucleotide and polynucleotide.
[0019] The term "gene" as used herein means a factor that defines inherited characteristics.
Usually, genes are arranged on a chromosome in a certain order. A gene that defines
the primary structure of protein is called a structural gene, and a gene that regulates
the expression of a structural gene is called a regulatory gene. In this specification,
the term "gene" sometimes refers to a "polynucleotide", "oligonucleotide" and "nucleic
acid".
[0020] The term "fragment" of a nucleic acid molecule as used herein refers to a polynucleotide
that is shorter than the entire length of the reference nucleic acid molecule, but
has a length sufficient for use as a factor of the present invention. Therefore, a
fragment as used herein is a polypeptide or a polynucleotide having a sequence length
of 1 to n-1 relative to the full length of a polypeptide or a polynucleotide (having
a length of n). The length of the fragment may be appropriately selected depending
on the purpose thereof, and a lower limit of length for a polypeptide can include
3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 and more amino acids. Lengths represented
by integers not specifically recited above (for example, 11) are also suitable as
a lower limit. Polynucleotides can include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40,
50, 75, 100 and more nucleotides. Lengths represented by integers not specifically
recited above (for example, 11) are also suitable as a lower limit.
[0021] The term "homology" of a gene as used herein represents the degree of identity to
each other between two ore more gene sequences. Therefore, the higher the homology
of two specific genes, the higher the identity and similarity of their sequences are.
Whether or not two selected genes have homology may be examined by a direct comparison
of their sequences, or hybridization under stringent conditions in the case of nucleic
acid sequences. During direct comparison of two gene sequences, typically, when at
least 50%, preferably at least 70%, more preferably, when at least 80%, 90%, 95%,
96%, 97%, 98% or 99% of the DNA sequences are identical between the two gene sequences,
the genes are determined to have homology.
[0022] Comparison of similarity and identity between base sequences and determination of
homology of base sequences are carried out herein by means of BLAST which is a sequence
analysis tool using default parameters.
[0023] "Expression" of a gene, polynucleotide, polypeptide or the like refers to the phenomenon
that the gene or the like takes a different form under certain circumstances in vivo.
Preferably, it means the phenomena of a gene, polynucleotide or the like being transcribed
and translated into a polypeptide. The phenomena of mRNA being produced as a result
of transcription may also be one form of expression. More preferably, the resultant
polypeptide may have undergone post-translational processing.
[0024] The term "a polynucleotide that hybridizes under stringent conditions" as used herein
refers to well-known conditions that are commonly used in the art. Such a polynucleotide
can be obtained by the colony hybridization method, the plaque hybridization method
or Southern blot hybridization using a polynucleotide selected from the polynucleotides
of the present invention as a probe. Specifically, it means a polynucleotide that
can be identified in the following manner. A filter on which DNA derived from colonies
or plaques is immobilized is used to carry out a polynucleotide hybridization in the
presence of 0.7-1.0 M NaCl at 65°C. Then, the filter is washed at 65°C with x0.1 to
x2 concentration of SSC (saline-sodium citrate) solution (150 mM sodium chloride,
15 mM sodium citrate). Hybridization may be conducted according to methods described
in laboratory manuals such as Molecular Cloning 2nd ed. , Current Protocols in Molecular
Biology, Supplement 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second
Edition, Oxford University Press (1995) and the like. Herein, "a sequence that hybridizes
under stringent conditions" preferably excludes sequences comprising exclusively A
or exclusively T.
[0025] The wording "hybridizable polynucleotide" refers to a polynucleotide that is able
to hybridize with other polynucleotides under the aforementioned hybridization conditions.
Specific examples of hybridizable polynucleotides include polynucleotides having at
least 60% or higher homology, preferably 80% or higher homology, more preferably 95%
or higher homology with a DNA base sequence encoding a polypeptide having the amino
acid sequence set forth in the SEQ ID NO:2, 4 or 6. As to the given homology, similarity
may be represented by a score, for example, by using the search program BLAST using
the algorithm developed by Altschul et al. (J. Mol. Biol. 215, 403-410 (1990)).
[0026] Amino acids may be denoted herein by the generally known three-letter coding or the
one-letter coding recommended by IUPAC-IUB Biochemical Nomenclature Commission. Likewise,
nucleotides may be denoted by the commonly accepted one-letter coding.
[0027] "Corresponding" amino acid used herein refers to an amino acids having or expected
to have a similar effect in a protein or polypeptide to the specific amino acid in
a protein or polypeptide which is the reference for comparison. In an enzyme molecule,
in particular, it means an amino acid which is located at a similar position in an
active site and similarly contributes to catalytic activity.
[0028] The term "nucleotide" as used herein may be naturally occurring or may not be naturally
occurring. The term "derivative nucleotide" or "nucleotide analogue" refers to a nucleotide
that is different from a naturally occurring nucleotide, but has a function similar
to that of the function of the original nucleotide. Such derivative nucleotides and
nucleotide analogues are well known in the art. Examples of such derivative nucleotides
and nucleotide analogues include, but are not limited to, phosphorothioate, phosphoroamidate,
methyl phosphonate, chiral methyl phosphonate, 2-O-methylribonucelotide and peptide-nucleic
acid (PNA).
[0029] The term"fragment" as used herein refers to a polypeptide or polynucleotide having
a sequence length of 1 to n-1 relative to the full length of a polypeptide or polynucleotide
(having a length of n). The length of the fragment may be appropriately selected depending
on the purpose thereof, and a lower limit of length for a polypeptide includes 3,
4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 and more amino acids. Lengths represented
by integers not specifically recited above (for example, 11) are also suitable as
a lower limit. For polynucleotides, fragment includes polynucleotides of 5, 6, 7,
8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more in length. Lengths represented
by integers not specifically recited above (for example, 11) are also suitable as
a lower limit.
[0030] The term "isolation (isolated)" as used herein refers to the state of a certain substance
or nucleic acid, and that the substance or nucleic acid is in a state that is different
from its naturally-occurring state, and that the substance or nucleic acid is not
accompanied by at least one substance or nucleic acid that accompanies it in its naturally-occurring
state. The term to "concentrate" a nucleic acid as used herein refers to increasing
the abundance of a specific nucleic acid compared to its naturally-occurring abundance.
Therefore, in a preferred condition, a concentrated nucleic acid or nucleic acid composition
contains only a specific nucleic acid.
[0031] The term to "purify" a specific substance as used herein refers to making the substance
in it's abundantly existing state, and reducing the concentration of substances other
than the specified substance to such a degree that they will not influence the function
of the specified substance. Therefore, in a preferred condition, a purified substance
or substance-containing composition contains only the specific substance.
[0032] In this specification the terms "purification (purified)" and "cloning (cloned)"
are interchangeably used. The terms "purification (purified)" and "cloning (cloned)"
refer to a state of a certain substance or nucleic acid, and refer to making the abundance
of the nucleic acid higher, preferably to the level that the substance or nucleic
acid is not substantially accompanied by other kinds of substances or nucleic acids.
When used in the contexts of "purification" and "cloning" herein, the term "state
where substantially no other kinds of substances or nucleic acids accompany" refers
to the state where these other kinds of substances or nucleic acids are completely
absent, or will not exert any influence on the substance or nucleic acid of interest
if present. Therefore, in a more preferred condition, a purified nucleic acid or nucleic
acid composition contains only a specific nucleic acid.
[0033] The term "purify" a specific substance as used herein refers to making the substance
in an abundantly existing state, and reducing the concentration of substances other
than the specified substance to such a degree that they will not influence the function
of the specified substance.
[0034] The term "gene transfer" as used herein refers to transferring a desired naturally-occurring,
synthetic or recombinant gene or gene fragment into a target cell in vivo or in vitro
in such a manner that the transferred gene maintains its function. A gene or gene
fragment transferred in the present invention encompasses DNA or RNA having a specific
sequence, or a nucleic acid which is a synthetic analogue thereof. The terms "gene
transfer", "transfection" and "transfect" as used herein are interchangeably used.
[0035] The terms "gene transfer vector" and "gene vector" as used herein are interchangeably
used. The terms "gene transfer vector" and "gene vector" refer to vectors capable
of transferring a polynucleotide sequence of interest into a target cell. Examples
of a "gene transfer vector" and "gene vector" include, but are not limited to, a "viral
envelope vector" and a "liposome vector".
[0036] The term "viral envelope vector" as used herein refers to a vector in which a foreign
gene is encapsulated in a viral envelope or a vector in which a foreign gene is encapsulated
in a component containing a protein derived from a viral envelope. The virus used
for preparing a gene transfer vector may be a wild-type virus or a recombinant virus.
[0037] In the present invention, examples of the virus used for preparing a viral envelope
or a protein derived from a viral envelope include, but are not limited to, viruses
belonging to families selected from the group consisting of Retroviridae, Togaviridae,
Coronaviridae, Flaviviridae, Paramyxoviridae, Orthomyxoviridae, Bunyaviridae, Rhabdoviridae,
Poxviridae, Herpesviridae, Baculoviridae and Hepadnaviridae. Preferably viruses belonging
to the family Paramyxoviridae, and more preferably HVJ (Hemagglutinating Virus of
Japan, Sendai Virus) are used.
[0038] Examples of proteins derived from a viral envelope include, but are not limited to
the F protein, HN protein, NP protein and M protein of HVJ.
[0039] The term "liposome vector" as used herein refers to a vector wherein a foreign gene
is encapsulated in a liposome. Examples of lipids used for preparing a liposome vector
include, but are not limited to, neutral phospholipids such as DOPE (dioleoyl phosphatidyl
ethanolamine) and phosphatidyl choline; negatively charged phospholipids such as cholesterol,
phosphatidyl serine and phosphatidic acid; and positively charged lipids such as DC-cholesterol
(dimethylaminoethanecarbamoyl cholesterol) and DOTAP (dioleoyl trimethylammonium propane).
[0040] The term "liposome" used herein is one type of lipid bilayer. For example, when a
phospholipid such as lecithin is suspended at 50% (by weight) or more in water at
a temperature higher than the gel-liquid crystal phase transition temperature which
is specific for said phospholipid, a closed vesicle composed of a lipid bilayer membrane
encapsulating a water phase is formed. This vesicle is called a liposome. Liposomes
are generally classified as multilayered liposomes (MLV: multilamellar vesicle), in
which a plurality of bilayer membranes overlap like an onion, and unilamellar liposomes
having only one membrane. The latter may also be prepared by making a suspension of
phospholipids such as phosphatidyl choline dispersed by intensive stirring with a
mixer, followed by an ultrasound treatment.
[0041] Liposomes having only one membrane are further classified into small single membrane
liposomes (SUV: small unilamellar vesicle) and large single membrane liposomes (LUV:
large unilamellar vesicle) according to their diameter. MLVs are prepared by adding
water to a lipid thin film and applying mechanical oscillation. SUVs may be prepared
by ultrasonication of MLVs or by removal of a surfactant from a mixture of lipid and
surfactant by dialysis or the like. Besides the above methods, other well-known methods
include (1) a method of preparing LUV by treating SUV with multiple freeze- thaw cycles;
(2) a method of preparing LUVs by fusing SUVs composed of acidic phospholipids in
the presence of Ca
2+ and then removing the Ca
2+ with EDTA (ethylenediamine tetraacetic acid), and (3) a method of preparing LUVs
and the like by causing phase conversion while distilling off ether from an emulsion
of lipids in solution with ether and water (reverse-phase evaporation vesicle: REV).
[0042] The terms "surfactant" and "surface activator" as used herein are interchangeably
used. A surfactant is a substance that exhibits strong surface-tension activity against
water, and forms an aggregate such as micelle in a solution at concentrations exceeding
the critical micelle concentration. A surfactant has both a hydrophilic moiety and
a hydrophobic (lipophilic) moiety , and strongly absorbs into a two-phase interface
of water and oil according to the balance of hydrophilicity and lipophilicity, to
significantly decrease the free energy (interface tension) at the interface. Typical
hydrophobic groups are long chain hydrocarbon groups such as alkyl groups; typical
hydrophilic groups may include ionic dissociation groups and nonionic polar groups
such as a hydroxyl group. Since surfactants having a carboxyl group, sulfo group,
hydrogen sulfate group, and -OSO-OH dissociate in water to become anions, they may
be called anionic surfactants. Typical examples include, but are not limited to, fatty
acid soaps, alkyl benzene sulfonate and the like. In contrast, those having a quaternary
ammonium group dissociate to become cations and are called cationic surfactants. There
are also surfactants having both a cationic dissociation group and an anionic dissociation
group in the same molecule, such as long chain alkyl amino acids, and such surfactants
are called amphoteric surfactants. Those surfactants having a nonionic polar group
are called nonionic surfactants, polyoxyethylenenonylphenyl ether is typical example
of a non-ionic surfactant.
[0043] The term "lipid" as used herein encompasses any lipids, as long as (1) they have
a long chain fatty acid or a similar hydrocarbon chain in the molecule, and (2) they
exist in an organism or they are molecules derived from an organism. Preferred lipids
are phospholipids capable of forming liposomes, and more preferred lipids include,
but are not limited to, phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol,
phosphatidyl ethanol amine, cholesterol, sphingomyelin and phosphatidic acid.
[0044] The term "fatty acid" as used herein refers to aliphatic monocarboxylic acids and
aliphatic dicarboxylic acids that are obtained by hydrolysis of naturally-occurring
lipids. Typical fatty acids include, but are not limited to, arachidonic acid, palmitic
acid, oleic acid and stearic acid.
[0045] The term "gene transfer activity" as used herein refers to the activity of "gene
transfer" by a vector, and may be detected using a function of the transferred gene
as an index indicator (for example, expression of the encoded protein and/or activity
of the protein, in the case of an expression vector).
[0046] The term "inactivation" as used herein refers to a virus with an inactivated genome.
Inactivated viruses are replication defective. Preferably, the inactivation is achieved
by UV treatment or treatment with an alkylation agent.
[0047] The term "foreign gene" as used herein refers to a nucleic acid contained in a viral
envelope vector but not originating from the virus, or a nucleic acid contained in
a liposome vector. In one aspect of the present invention, the foreign gene is operatively
linked with a regulatory sequence which allows the gene transferred by a gene transfer
vector to be expressed (e.g., a promoter, enhancer, terminator and a poly A addition
signal are required for transcription, and a ribosome binding site, initiation codon
and a termination codon are required for translation). In another aspect of the present
invention, the foreign gene does not include a regulatory sequence for expression
of the foreign gene. In a further aspect of the present invention, the foreign gene
is an oligonucleotide or a decoy nucleic acid.
[0048] A foreign gene contained in a gene transfer vector is typically a nucleic acid of
DNA or RNA, and the transferred nucleic acid molecule may include a nucleic acid analogue
molecule. Molecular species contained in a gene transfer vector may be a single gene
molecule species or a plurality of different gene molecule species.
[0049] The term "gene library" as used herein means a nucleic acid library including nucleic
acid sequences isolated from the natural world or synthetic nucleic acid sequences.
Examples of the source of nucleic acid sequences isolated from the natural world,
include, but are not limited to, genome sequences and cDNA sequences derived from
eukaryotic cells, prokaryotic cells, or viruses. A library of sequences isolated from
the natural world to which optional sequences (e.g., signal sequences or tag sequences)
are added is also encompassed in the gene library of the present invention. In one
embodiment, a gene library also includes sequences such as promoter sequences that
enable transcription and/or translation of the nucleic acid sequences contained in
the library.
[0050] The terms "HVJ" and "Sendai Virus" as used herein are used interchangeably. For example,
the terms "envelope of HVJ" and "envelope of Sendai Virus" are synonymously used herein.
"Sendai Virus" as used herein belongs to the genus paramyxovirus in the family Paramyxoviridae,
and has cell fusion activity. The viral particles are enveloped, and are polymorphic
in that the particle diameter varies from 150 to 300 nm. The genome is a (-) strand
RNAmolecule having a length of about 15500 bases. The virus has an RNA polymerase,
is thermally unstable, hemagglutinates almost all types of erythrocyte and exhibits
hemolytic activity.
[0051] The term "HAU" used herein refers to a measure of virus activity that is able to
hemagglutinate 0.5% of chicken erythrocytes, wherein 1 HAU corresponds to about 24
million viral particles (Okada, Y. et al., Biken Journal 4, 209-213, 1961).
[0052] The term "candidate nucleic acid" as used herein may be any nucleic acid insofar
as it is an obj ect to be purified. Herein, a population of candidate nucleic acids
may be obtained directly from cells, a first host cell such as a mammalian cell as
a source, or obtained as an isolated nucleic acid preparation. The population of candidate
nucleic acids thus obtained is purified using a second host cell.
[0053] Examples of animal cells that may be used as a host cell include, but are not limited
to, mouse myeloma cell lines, rat myeloma cell lines, mouse hybridoma cells, CHO cells
which are derived from the Chinese hamster, BHK cells, African green monkey kidney
cell lines, human leucocyte-derived cell lines, the cell line HBT5637 (Japanese Laid-Open
Publication No. 63-299) and human colon cancer cell lines. Mouse myeloma cell lines
include, ps20, NSO and the like. Rat myeloma cell lines include YB2/0 or the like.
Human embryo kidney cell lines include HEK293 (ATCC: CRL-1573) or the like. Human
leucocyte-dervied cell lines include BALL-1 or the like. African green monkey kidney
cell lines include COS-1, COS-7 or the like. Human colon cancer cell lines include
HCT-15 or the like.
[0054] The term "animal" as used herein is used in the broadest sense within the art and
includes vertebrates and invertebrates. Examples of animals include, but are not limited
to, the classes Mammalia, Aves, Reptilia, Amphibia, Pisces, Insecta, Vermes and the
like.
[0055] The term "tissue" of an organism as used herein refers to a population of cells having
a certain similar ability across the population. Therefore, the tissue may be a part
of an organ. A particular organ often has cells having the same function, however,
it may include cells having slightly different functions. Therefore, in this specification,
a variety of cells may be included in a particular tissue insofar as they commonly
have a certain characteristic.
[0056] The second host cell is not particularly limited insofar as it is a cell capable
of "gene-transferring" a candidate nucleic acid; various host cells that are conventionally
used in genetic engineering (for example, prokaryotic cells and eukaryotic cells)
may be used.
[0057] Examples of prokaryotic cells include, prokaryotic cells belonging to the genus selected
from the group consisting of Escherichia, Bacillus, Streptococcus, Staphylococcus,
Haemophilus, Neisseria, Actinobacillus, Acinetobacter, Serratia, Brevibacterium, Corynetbacterium,
Microbacterium, and Pseudomonas, for example, Escherichia coli XL1-Blue, Escherichia
coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276,
Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia
coli No. 49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli BL21
(DE3), Escherichia coli BL21 (DE3) pLysS, Escherichia coli HMS174 (DE3), Escherichia
coli HMS174 (DE3) pLysS, Serratia ficaria, Serratia fonticola, Serratia liquefaciens,
Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium
ammmoniagenes, Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticum
ATCC14066, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067,
Corynebacterium glutamicum ATCC13869, Corynebacterium acetoacidophilum ATCC13870,
Microbacterium ammoniaphilum ATCC15354, Pseudomonas sp D-0110 and the like.
[0058] Examples of eukaryotic cells include, yeast strains belonging to the genus Saccharomyces,
Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichia, and fungi
belonging to Neurospora, specifically, Saccharomyces cerevisiae, Schizosaccharomyces
pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia
pastoris and the like. As a method of transferring a recombinant vector in the host
cells, any method for transferring DNA into fungi can be used, such methods include
electroporation methods [Methods Enzymol., 194, 182 (1990)], spheroplast-based methods
[Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], lithium acetate-based methods [J. Bacteriol.,
153, 163 (1983)] and the method described in Proc. Natl. Acad. Sci. USA, 75, 1929
(1978).
[0059] Plant cells, for example, include plant cells derived from potato, tobacco, corn,
rice plant, rapeseed, soy bean, tomato, carrot, wheat, barley, rye, alfalfa and flax.
As a method of transferring a recombinant vector, any method for transferring DNA
into a plant cell can be used. Examples of such methods include the use of Agrobacterium
(Japanese Laid-Open Publication No. 59-140885, Japanese Laid-Open Publication No.
60-70080, WO94/00977), electroporation methods (Japanese Laid-Open PublicationNo.
60-251887) andmethodsusingaparticlegun (gene gun) (Japanese patent No. 2606856, Japanese
patent No. 2517813) .
[0060] Insect cells may include Spodoptera frugiperda ovary cells, Trichopllusia ni ovary
cells, cultured cells derived from silkworm ovaries and the like. Examples of Spodoptera
frugiperda ovary cells include Sf 9, Sf21 (Baculovirus Expression Vectors: A Laboratory
Manual) and the like, examples of Trichopllusia ni ovary cells include High 5, BTI-TN-5B
1-4 (Invitrogen) and the like, and examples of the culture cells derived from silkworm
ovaries include Bombyx mori N4.
[0061] The term "variant" as used herein refers to substances which are partially modified
from the original substances such as polypeptides or polynucleotides. A variant includes
substitution variants, addition variants, deletion variants, truncated variants, allelic
mutants and the like. Alleles are genetic variants which belong to the same locus
but are distinct from each other. Therefore, the term "allelic gene mutant" means
a variant which forms an allelic relative to a certain gene. The term "species homolog
or homolog" denotes to that the substance has a homology (preferably, homology of
60% or more, more preferably, homology of 80% or more, 85% or more, 90% or more, 95%
or more) with a certain gene in a certain species at the amino acid level or nucleotide
level. A method for obtaining such a species homolog is apparent from the description
of this specification. An "ortholog" is also called an "orthologous gene", the term
is used for two genes that result from speciation of a specific common ancestor. Taking
the hemoglobin gene family having a multigene structure as an example, human and mouse
α-hemoglobin genes are orthologs, but the human α-hemoglobin and β-hemoglobin genes
are paralogs (genes generated as a result of gene duplication). Since orthologs are
useful for estimating a molecular phylogenetic tree, orthologs may also be useful
in the present invention.
[0062] The term "conservative (conservatively modified) variant" is applicable both to amino
acid sequences and nucleic acid sequences. Regarding a specific nucleic acid sequence,
a conservative variant refers to a nucleic acid that encodes the same or substantially
the same amino acid sequence. When the nucleic acid does not encode an amino acid
sequence, it refers to substantially the same sequence. Because of the degeneracy
of genetic codes, a large number of functionally equivalent nucleic acids encode any
particular protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino
acid, alanine. Therefore, at every position where a codon specifies alanine, the codon
may be changed into any corresponding codon described above without changing the encoded
polypeptide. Such variation of nucleic acids is "silent variant (mutation)" which
is one of the conservative variants. Any nucleic acid sequence encoding a polypeptide
herein also describes all possible silent mutations for the nucleic acid. One skilled
in the art will recognize that each codon in a nucleic acid (excluding AUG which is
usually a unique codon for methionine, and TGG which is usually a unique codon for
tryptophan) may be modified so as to produce a functionally identical molecule. Therefore,
all possible silent mutations of a nucleic acid encoding a polypeptide is implied
in each of the described sequences. Preferably, such modification may be made so as
to avoid substitution of cysteine which is an amino acid that exerts great influence
on the higher structure of polypeptides.
[0063] In order to create a functionally equivalent polypeptide, addition, deletion or modification
of amino acid may be carried out herein besides substitution of amino acid. Substitution
of an amino acid refers to making a substitution in the original peptide with at least
one amino acid, for example 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino
acids. Addition of an amino acid refers to adding onto the original peptide, at least
one amino acid, for example 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino
acids. Deletion of an amino acid refers to deleting from the original peptide, at
least one amino acid, for example 1 to 10, preferably 1 to 5, more preferably 1 to
3 amino acids. Examples of amino acid modification include, but are not limited to,
amidation, carboxylation, sulfation, halogenation, alkylation, glycosylation, phosphorylation,
hydroxylation and acylation (for example, acetylation). Amino acids that are substituted
or added may be naturally occurring amino acids, non-naturally occurring amino acids,
or amino acid analogues. Naturally occurring amino acids are preferred.
[0064] Such a nucleic acid may be obtained using well-known PCR methods or chemical synthesis.
Site-directed mutagenesis, hybridization methods and the like may be combined with
the methods described above.
[0065] The term "substitution, addition or deletion" of a polypeptide or polynucleotide
as used herein refers to the occurrence of substitution, addition or deletion of an
amino acid or it's alternative, or a nucleotide or it's alternative from the original
polypeptide or polynucleotide. Techniques for substitution, addition or deletion are
well known in the art, and include, for example, site-directed mutagenesis and the
like. The number of substitutions, additions or deletions is at least one, and any
number is acceptable insofar as a function of interest (for example, a cancer marker,
a neurological disease marker or the like) is retained in a variant having such a
substitution, addition or deletion. For example, the number may be one or several,
preferably within 20%, within 10% of the entire length, or less than or equal to 100,
less than or equal to 50, or less than or equal to 25.
[0066] General molecular biological methods that may be used herein can be readily practiced
by a person skilled in the art with reference to, for example, Ausubel F.A. et al.
ed. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; SambrookJ.
et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY).
[0067] The term "expression plasmid" as used herein refers to a nucleic acid sequence in
which, in addition to a structural gene and a promoter for regulating expression thereof,
various regulatory elements are linked so as to be able to operate in a host cell.
Preferably, the regulatory elements may include a promoter, a terminator and a selection
marker. It is well-known by those skilled in the art that the type of expression plasmid
and the kind of regulatory element used may vary depending on the host bacterial cell.
In the present invention, an expression plasmid expresses a candidate nucleic acid
in a first host cell and/or second host cell. Therefore, the expression plasmid includes
a candidate nucleic acid, and a regulatory element (for example, promoter) operably
linked with the candidate nucleic acid.
[0068] The term "promoter" used herein refers to a region of DNA that determines the transcription
initiation site of a gene and directly regulates the frequency of transcription thereof,
and a base sequence where RNA polymerase binds to start transcription. A putative
promoter region varies with the particular structural gene, and is usually positioned
upstream of a structural gene. Not limited to these, a putative promoter may also
be positioned downstream of a structural gene.
[0069] A promoter may be inducible, constitutive, site specific, or period specific. As
the promoter, any promoters that can be expressed in a host cell such as mammalian
cells, Escherichia coli, yeast or the like are acceptable.
[0070] When used herein concerning the expression of a gene, the term "site specificity"
generally refers to the specificity of expression of the gene within specific mammalian
tissues. The term "period specificity" refers specifically to expression of the gene
in accordance with the developmental stage of a mammal. Such specificity may be introduced
into a desired organism by selecting an appropriate promoter.
[0071] When the expression of a promoter of the present invention is described as "constitutive"
herein, it means that in all tissues in organism, the gene is expressed in an almost
constant amount regardless of the growth/proliferation of the organism. More specifically,
if during Northern blotting analysis almost the same level of expression is observed
both in the same or in a corresponding site at given points (for example, two or more
points (for example, at 5 days, and 15 days)) the expression is said to be constitutive
according to the definition of the present invention. Constitutive promoters are believed
to play a role in maintaining the homeostasis of organisms in normal growth environments.
The term that the expression of a promoter of the present invention is "responsive"
means that when at least one factor is given to an organism, the level of expression
changes. In particular, when expression levels increase in response to at least one
factor the expression is said to be "inducible" by the factor, and when expression
levels reduce in response to at least one factor the expression is said to be "reductive"
by the factor. "Reductive" expression is based on the premise the fact that expression
is initially observed under normal conditions, and hence "reductive" expression is
concept that overlaps "constitutive" expression. These properties may be determined
by analyzing RNA extracted from a specific tissue of an organism in order to analyze
the expression levels by Northern blotting analysis or by quantitating the expressed
protein by Western blotting. A mammalian cell or a mammal (including a specific tissue)
transformed with a vector that incorporates a promoter inducible by a factor, as well
as a nucleic acid encoding a site specific recombinant inducing factor of the present
invention, may be subjected to site specific recombination of the site specific recombination
sequence under certain conditions by using a stimulating factor having the function
of inducing the promoter.
[0072] As a potent promoter for expression in a mammalian cell, a variety of naturally occurring
promoters (for example, the early promoter of SV40, the EIA promoter of adenovirus,
the promoter of human cytomegarovirus (CMV), the human elongation factor-1 (EF-1)
•promoter, the promoter of the Drosophila minimum heat shock protein 70 (HSP),the
human metallothionein (MT) promoter, the Rous-sarcoma virus (RSV) promoter, the human
ubiquitinC (UBC) promoter, human act in promoter), and artificial promoters (for example,
fusion promoters such as SRα promoter (a fusion of the SV40 early promoter and the
LTR promoter of HTLV) and the CAG promoter (a hybrid of the CMV-IE enhancer and the
chicken act in promoter)) are well known. Therefore, by using these well-known promoters
or variants thereof, it is possible to readily increase the expression level .
[0073] When Escherichia coli is used as a host cell, promoters derived from Escherichia
coli or phages such as the trp promoter (Ptrp), the lac promoter (Plac), the PL promoter,
the PR promoter, the PSE promoter, the SPO1 promoter, the SPO2 promoter, the penP
promoter and the like can be exemplified. Also artificially designed and modified
promoters such as a promoter comprising two serially linked Ptrps (Ptrp x2), the tac
promoter, the lacT7 promoter, the let I promoter and the like may be used.
[0074] The term "enhancer" may be used herein for improving the expression efficiency of
a gene of interest. A typical enhancer when used in a mammalian cell includes, but
is not limited to, an enhancer of SV40. An enhancer may be used singly or in plural,
or may not be used at all.
[0075] The term "terminator" as used herein refers to a sequence that is positioned downstream
of a protein coding region of a gene, and is involved in the termination of transcription
and the addition of a poly A tail when DNA is transcribed into mRNA. A terminator
is known to be involved in the stability of mRNA and to influence the expression level
of a gene.
[0076] The term "operably linked" as used herein means that expression (operation) of an
intended sequence is placed under the control of a transcription and translation regulatory
sequence (for example, a promoter, an enhancer or the like) or under a translation
regulatory sequence. In order to operably link a promoter to a gene, usually, the
promoter is located directly upstream of the gene, however, it is not necessarily
located adjacently.
[0077] As used herein, the term "biological activity" is used interchangeably with the term
"functional property". The terms "biological activity" and "functional property" as
used herein mean an activity that a certain factor (for example, a nucleic acid) may
have in an organism, and encompasses activities exerting various functions. For example,
when a certain factor is a gene encoding a growth factor, the functional property
encompasses expressing the growth factor in a host cell, and preferably promoting
the growth of the cell by expression of the growth factor. For example, when the certain
factor is a gene encoding an enzyme, the functional property encompasses expressing
the enzymatic activity in a host cell and preferably increasing enzymatic activity
to a detectable level. In another example, when the certain factor is a gene encoding
a ligand, the functional property encompasses expressing a ligand that binds a receptor
corresponding to the ligand, and preferably changes the phenotype of a cell having
the receptor corresponding to the ligand, by the expression of the ligand.
[0078] In this specification, when it is necessary to transfer a nucleic acid into a second
host cell, anymethod for transferring DNA into a host cell canbe used. Examples of
such methods include, transfection, transduction, transformation (for example, electroporation
, methods using a particle gun (gene gun) and the like).
[0079] When referring to gene herein, the term "expression plasmid" means a nucleic acid
capable of expressing a gene included in a polynucleotide sequence of interest after
the polynucleotide sequence of interest is transferred into a cell of interest. Expression
plasmids include plasmids having a promoter at a position suited for transcription
of the polynucleotide to be expressed.
[0080] In this specification, "detection" or "quantification" of the expression of a nucleic
acid transferred into a host cell may be achieved by using appropriate methods including
measurement of mRNA and immunological measuring methods. Examples of molecular biological
measuring methods include Northern blotting, Dot blotting, PCR and the like. Examples
of immunological measuring methods include ELISA using a micro titerplate, RIA, fluorescent
antibodymethods, Westernblotting, immunohistochemical staining and the like. Quantification
methods include ELISA or RIA.
[0081] In this specification, "detection" or "quantification" of expression of nucleic acids
transferred into a host cell may be carried out using a solid phase (for example,
a substrate, support, array, chip or microchip).
[0082] The terms "substrate" and "support" are used in the same meaning herein, and refer
to a material (preferably solid) from which an array of the present invention is constructed.
A material for the substrate includes any solid material having the characteristic
of binding a biological molecule used in the present invention via a covalent or noncovalent
bond or any material that can be derivatized to have such a characteristic.
[0083] Materials used for a substrate include any material capable of forming a solid surface,
and include, but are not limited to, for example, glass, silica, silicones, ceramics,
silicon dioxide, plastics, metals (including alloy), naturally occurring or synthetic
polymers (for example, polystyrene, cellulose, chitosan, dextran and nylon). A substrate
may have a plurality of layers formed of different materials. For example, inorganic
insulating materials, such as glass, quartz glass, alumina, sapphire, forsterite,
silicon carbide, silicon oxide, silicon nitride and the like may be used. Also organic
materials such as polyethylene, ethylene, polypropyrene, polyisobutylene, polyethylene
terephtalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride,
polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic
resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol
resin, urea resin, epoxy resin, melamine resin, styrene-acrylonitrile copolymer, acrylonitrile-butadiene-styrene
copolymer, silicone resin, polyphenylene oxide, polysulfone and the like may be used.
In the present invention, membranes that are used for blotting such as nylon membrane,
nitrocellulose membrane, PVDF membrane and the like may be used. A nylon membrane
is preferred because the result may be analyzed using a convenient analyzing system
when a nylon membrane is used. However, when an object of high density is analyzed,
the use of hard materials such as glass is preferred.
[0084] The term "chip" or "microchip" as used herein refers to an ultrasmall integrated
circuit which has a plurality of functions and forms a part of a system. As used herein,
a "DNA chip" includes a substrate and DNA, and at least one DNA molecule (for example,
cDNA fragment) is placed on the substrate. As used herein, a "protein chip" includes
a substrate and protein, and at least one protein (for example, a polypeptide or oligopeptide)
is placed on the substrate. As used herein, a "DNA chip" and a "protein chip" are
encompassed by the terms "microchip" or simply "chip". The term "microarray" means
a chip on which at least one biological molecule (for example, an oligonucleotide
such as a cDNA fragment, or a peptide) is placed in array.
[0085] A biological molecule (for example, an oligonucleotide such as a cDNA fragment or
a peptide) as used herein may be collected from an organism ormaybe chemically synthesized
using methods known by those skilled in the art. For example, oligonucleotides may
be prepared by automated chemical synthesis using either a DNA synthesizer or a peptide
synthesizer commercially available from Applied Biosystems or the like. Compositions
and methods for automated oligonucleotide synthesis are disclosed in, for example,
USP No. 4,415,732, Caruthers et al. (1983) ; USP No. 4, 500, 707 and Caruthers (1985);
USP No. 4,668,777, Caruthers et al. (1987).
[0086] On a substrate, any number of biological molecules (for example, DNA molecules or
peptides) may be placed, and usually up to 10
8 biological molecules, and in another embodiment, up to 10
7 biological molecules, up to 10
6 biological molecules, up to 10
5 biological molecules, up to 10
4 biological molecules, up to 10
3 biological molecules, or up to 10
2 biological molecules may be placed on one substrate. In these cases, the size of
the substrate is preferably as small as possible. In particular, the size of a spot
of a biological molecule (for example, a DNA molecule or a peptide) may be as small
as the size of a single biological molecule (i.e., in the order of 1-2 nm). In some
cases, the minimum substrate area is determined by the number of biological molecules
on the substrate.
[0087] The term "biological molecule" as used herein refers to molecules associated with
organisms. As used herein, the term "organisms" refers to a biological organism including,
but not limited to, animals, plants, fungi and viruses. The term "biological molecules"
encompasses molecules extracted from organisms, however, is not limited to this, and
any molecule that influences an organism is encompassed by the definition of a biological
molecule. Examples of such biological molecules include, but are not limited to, proteins,
polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides,
nucleic acids (including, for example, DNA molecules such as cDNA and genomic DNA,
and RNA molecules such as mRNA, polysaccharides, oligosaccharides, lipids, small molecules
(for example, hormones, ligands, signal transducers, organic small molecules and the
like), and composite molecules thereof. Asused herein,biologicalmoleculesmay be preferably
peptides, DNA or RNA.
[0088] In the case where a host cell changes due to a nucleic acid transferred into the
host cell, it is possible to "detect" or "quantify" the expression of the nucleic
acid transferred into the host cell by measuring the degree of the change. Examples
of such changes in host cells include, but are not limited to, changes in enzymatic
activity of a specific enzyme in a cell, changes in cell growth rate, and the like.
[0089] The term "expression amount" refers to the amount in which a polypeptide or mRNA
is expressed in a cell of interest. Such an expression amount may be the level of
protein expression of the polypeptide of the present invention evaluated by any appropriate
method, including immunological measuring methods such as ELISA, RIA, fluorescent
antibodies, Western blotting and immunohistochemical staining using a antibody of
the present invention; or the level of mRNA expression of the polypeptide of the present
invention evaluated by any appropriate methods, including molecular biological methods
such as Northern blotting, dot blotting, and PCR and the like. As used herein, "expression
amount" may be an absolute value represented by a numerical unit, such as expressed
weight, absorbance having correlation with expressed weight and the like, or may be
a relative value represented by a ratio relative to a control or comparison reference.
"Change in expression amount" means increase or decrease in the expression amount
in the protein level or in the mRNA level of the polypeptide of the present invention
as evaluated by any appropriate method including the forgoing immunological or molecular
biological methods.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0090] In the present invention, a method of isolating a nucleic acid having an intended
functional property from candidate nucleic acids can be carried out by the following
steps:
(A) transferring a nucleic acid into a plurality of first host cells and allowing
the nucleic acid to transiently express therein;
(B) selecting, from the plurality of first host cells into which the nucleic acid
is transferred, a cell having the nucleic acid of the intended functional property
transferred therein;
(C) preparing a purified nucleic acid from the selected cell; and
(D) selecting a purified nucleic acid having the intended functional property.
[0091] In the above method, the candidate nucleic acids may be derived from any organism,
and may be DNA, RNA or nucleotide analogues. The candidate nucleic acids may be one
kind or a plurality of kinds.
[0092] In the above methods, the first host cells are preferably animal cells, more preferably
mammalian cells including human cells, although not particularly limited thereto.
The candidate nucleic acids may be transferred into the first host cells using a variety
of known methods. Typical methods include, but are not limited to, a transferring
method using a viral envelope, a transferring method using a liposome, a transferring
method using a liposome containing at least one protein from a viral envelope, a transferring
method using calcium phosphate, and an electroporation method. When the transferring
method using a viral envelope or the transferringmethodusing a liposome containing
at least one protein from a viral envelope is used, the virus used for preparing the
viral envelope may be derived from viruses belonging to a family selected from the
group consisting of Retroviridae, Togaviridae, Coronaviridae, Flaviviridae, Paramyxoviridae,
Orthomyxoviridae, Bunyaviridae, Rhabdoviridae, Poxviridae, Herpesviridae, Baculoviridae
and Hepadnaviridae. Preferably, the virus is viruses of the family Paramyxoviridae,
particularly HVJ.
[0093] A method of transferring candidate nucleic acids into first host cells using a liposome
containing at least one protein from viral envelope is also available. Examples of
the protein used in this method include, but are not limited to, F protein, HN protein,
NP protein, M protein, or a combination thereof.
[0094] The methods for transferring the candidate nucleic acids into first host cells are
also applicable to second host nucleic acids and third host nucleic acids.
[0095] Moreover, after transferring candidate nucleic acids into first host cells, mutation
may be induced in the first host cells to increase the diversity of the nucleic acids
transferred in the host cells. Mutagenesis methods for cells are well known, and examples
of such methods include, but are not limited to, methods using chemicals such as ethidium
bromide and nitrosoguanidine, and physical methods such as UV irradiation, X-ray irradiation
and radioactive ray radiation. In the present invention, after increasing the diversity
through mutagenesis, a candidate nucleic acid having the intended functional property
may be selected from the resultant mutants.
[0096] When candidate nucleic acids are expressed in the first host cells, the candidate
nucleic acids may be operably linked with a regulatory element such as promoter. This
expression may be transient or stable.
[0097] Transient expression of candidate nucleic acids in the first host cells is sufficient.
However, the host cells may allow stable expression of the candidate nucleic acids
after the transient expression of candidate nucleic acids occurs. Such a host cell
is also within the scope of the present invention.
[0098] Next, from the host cells into which the nucleic acids are transferred, a cell into
which a nucleic acid having the intended functional property has been transferred
is selected. The intended functional property is selected, for example, from the group
consisting of induction of angiogenesis, tumor suppression, enhancement of osteogenesis,
induction of apoptosis, cytokine secretion, induction of dendrites, suppression of
arteriosclerosis, suppression of diabetes; suppression of autoimmune diseases; suppression
of Alzheimer's disease, suppression of Parkinson's disease, protection of nerve cells
and combinations thereof
[0099] Preferably, this selection is effected based on the phenotype of a host cell which
changes in response to expression of candidate nucleic acids. For example, when a
gene encoding a growth factor is isolated from candidate nucleic acids, the intended
functional property is promotion of growth of a specific cell or any cells.
[0100] The method of the present invention may be applicable to any functional properties
as long as the functional property of interest is recognizable. Examples of functional
properties intended in the present invention include, but are not limited to, promotion
or suppression of cell growth; differentiation or de-differentiation of cells; expression
of marker proteins or suppression of the expression of marker proteins; expression
of marker mRNA or suppression of expression of marker mRNA; change in membrane potential;
depolarization; apoptosis; carcinogenesis; arrest of growth; change in morphology;
change in size and the like.
[0101] Regarding the method for preparing a purified nucleic acid from a cell that is selected
as containing a nucleic acid having the intended functional property, the second host
cell may or may not be used. When the second host cell is used, the method is carried
out in the following steps without limitation:
(i) extracting a nucleic acid from the selected cell;
(ii) transferring the extracted nucleic acid into a second host cell to thereby obtain
a transformed cell;
(iii) purifying the transformed cell; and
(iv) preparing a nucleic acid from the purified, transformed cell.
[0102] In the foregoing method, a variety of well-known methods and commercially available
kits may be used to extract nucleic acids from the first host cells. Depending on
the kind of the second host cell, various well-known methods may be applied so as
to transfer the nucleic acid to the second host cell. For example, when a bacterium
is used as the second host cell, gene transfer may be achieved in the following manner
without any limitation: preparing competent cells by a calcium method or the like,
and transferring nucleic acids into bacterial host cells by the application of heat
shock. Electroporation may also be used. Non-limiting examples of methods for transferring
DNA into fungi include the use of electroporation [Methods. Enzymol., 194, 182 (1990)],
spheroplasts [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], and lithium acetate. When
a plant cell is used as a host cell, known methods include, but are not limited to,
the use of Agrobacterium (Japanese Laid-Open Publication No. 59-140885, Japanese Laid-Open
Publication No. 60-70080, WO94/00977), electroporation methods (Japanese Laid-Open
PublicationNo. 60-251887) and methods using a particle gun (gene gun). When an animal
cell is used as a host cell, available methods include but are not limited to, a transferring
method using a viral envelope, a transferring method using a liposome, a transferring
method using a liposome containing at least one protein from viral envelope, a transferring
method using calcium phosphate, and electroporation methods.
[0103] In the case where a second host cell is used, preferably only one kind of candidate
nucleic acid per cell is transferred into the second host cell. Therefore, when a
population of candidate nucleic acids is obtained, the population is transferred into
the second host cell, and a host cell having a nucleic acid transferred therein is
purified, whereby the candidate nucleic acid can be purified.
[0104] If a plurality of kinds of purified nucleic acids are observed after purification
of the nucleic acids, a purified nucleic acid having the intended functional property
may be selected from the purified nucleic acids. This selection may be achieved by
expressing the purified nucleic acid, confirming the function of the nucleic acid,
and determining whether or not the confirmed function is the intended function. Alternatively,
it may be achieved by sequencing the whole or a part of the purified nucleic acid
structure.
[0105] Kits for practicing the methods of the present invention are also provided in the
present invention.
(1. Preparation of a viral envelope vector)
[0106] Various methods for preparing a viral envelope vector are known in the art. For example,
the present inventors developed a hybrid gene transfer vector by combining a viral
vector and a non-viral vector, and constructed a fusion forming viral liposome having
a fusion forming envelope derived from hemagglutinating virus of Japan (HVJ: Sendai
Virus) (Kaneda, BiogenicAmines, 14:553-572 (1998) ; Kaneda et al, Mol. Med. Today,
5:298-303 (1999)). In this delivery system, a liposome filled with DNA is fused with
UV inactivated HVJ, to thereby form a HVJ liposome which is a fusion forming viral
liposome (diameter: 400-500 nm). Fusion-mediated delivery is advantageous in that
transfection of DNA is protected from endosomal degradation and lysosomal degradation
in the recipient cell. DNA having a length of up to 100 kb is incorporated into an
HVJ liposome, and delivered into a mammalian cell. RNA, oligonucleotides and drugs
are also introduced into a cell efficiently in vitro or in vivo. HVJ-liposome was
not shown to induce significant cell damage in vivo.
[0107] Repeated transfection in vivo has succeeded due to the low immunogenicity of HVJ
(Hirano et al., Gene Ther. , 5: 459-464 (1998)). This vector system was modified,
and anion type and cation type HVJ-liposomes have been developed for more efficient
gene delivery (Saeki at al., Hum. Gene Ther., 8:1965-1972 (1997)). Using this HVJ-liposome
system, a great number of gene therapy strategies have succeeded (Dzau et al, Proc.
Natl. Acad. Sci. USA, 93:11421-11425(1996); Kaneda et al., Mol. Med. Today, 5:298-303
(1999)). Several attempts to construct HVJ-derived synthetic virosomes have also been
made (Wu et al., Neuroscience Lett., 190:73-76 (1995)); Ramanietal., FEBSLett., 404:164-168
(1997) ; Ramani et al. , Proc. Natl. Acad. Sci. USA, 95:11886-11890 (1998)).
[0108] When it is necessary to inactivate a virus for preparing a vector, a variety of known
methods may be used. Typical inactivation methods include, but are not limited to,
UV irradiation, treatment with an alkylation agent, treatment with β-propiolacton,
treatment with surfactant, and partial degradation of the envelope by enzymatic treatment.
[0109] Modifications to the foregoing methods of preparing a viral envelope vector are also
known. Typical examples are shown below. The method of preparing a viral envelope
vector shown below is only for illustration, and the present invention is not limited
to vectors that are prepared in the method described below.
(1.1. Preparation of a gene transfer vector encapsulating a foreign gene in a component
containing a protein derived from viral envelope)
[0110] Examples of a gene transfer vector containing a protein derived from a viral envelope
include, but are not limited to, gene transfer vectors consisting of liposomes obtained
by reconstitution of the F fusion protein and HN fusion protein of HVJ (Sendai Virus),
but not including an amount of HVJ genomic RNA that is detectable by RT-PCR.
[0111] The F fusion protein and HN fusion protein used in preparation of such a gene transfer
vector may be protein of naturally-occurring HVJ or recombinantly expressed protein.
Recombinantly produced fusion proteins are subjectedto in vitro processing with proteases,
or processing with endogenous proteases in a mammalian host cell.
[0112] A gene transfer vector containing a protein derived from a viral envelope is prepared,
for example, by a method comprising the following steps:
- isolating a fusion protein from HVJ virus that has not been irradiated with UV;
- reconstituting the fusion protein in the presence of a surfactant and a lipid to prepare
a reconstituted particle;
- preparing a liposome filled with the intended nucleic acid; and
- fusing the reconstituted particle and the liposome.
[0113] The surfactant used in the above method is not particularly limited to a specific
surfactant, and preferred examples include octylglucoside, Triton-X100, CHAPS or NP-40,
or mixtures thereof.
[0114] The lipid used in the above method is not particularly limited to a specific lipid,
and may be those (1) having a long chain fatty acid or a similar hydrocarbon chain
in the molecule, and (2) naturally occurring in an organism or derived from an organism.
Preferred examples of the lipid include, but are not limited to, phosphatidyl choline,
phosphatidyl serine, cholesterol, sphingomyelin, and phosphatidic acid.
[0115] A preparation method of liposomes is well known, and for example, the following method
may be used:
(A) Prepare a thin film of phospholipids in a first test tube in advance. Nitrogen
gas saturated with water at 55°C is introduced to this test tube and allowed to sufficiently
hydrate the thin film of phospholipids. Upon completion of hydration, the thin film
turns transparent.
(B) To the test tube A, a buffer of from a second test tube is added smoothly, and
after introduction of nitrogen gas, the test tube is sealed (for example, with Parafilm)
and kept in an incubator (which is an apparatus that enables the experiment to be
conducted at a constant temperature) at 37°C for about two hours.
(C) Macro-liposomes are prepared by gentle shaking. As a result of generation of liposomes,
the liquid becomes slightly cloudy. Using this, the following measurement is conducted.
(D) Placing a droplet of sample on a slide glass, the morphology of the liposomes
is observed under a fluorescence microscope (x1000).
[0116] A method of preparing a protein that is required for preparing a gene transfer vector
encapsulating a foreign gene in a component containing a protein derived from a viral
envelope is well known.
[0117] For example, it may be prepared by purification of HVJ envelope protein from naturally
occurring HVJ, or purification of recombinantly expressed HVJ envelope protein. Examples
of well-known protein purification methods include, but are not limited to, ammonium
sulfate precipitation, electrofocusing, and purification using a column. When a protein
is purified using a column, various columns may be selected depending on the properties
of the intended protein and the properties of likely contaminants. Examples of columns
used for protein purification include, but are not limited to, an anion exchange column,
a cation exchange column, a gel filtration column and an affinity column.
[0118] Alternatively, a gene transfer vector containing a protein derived from a viral envelope
is prepared by a method comprising the steps of:
- recombinantly expressing the F protein and HN protein of HVJ;
- processing F protein with a protease;
- isolating F protein and HN protein;
- reconstituting F protein and HN protein in the presence of a surfactant and lipids
to prepare a reconstituted particle;
- preparing a liposome filled with nucleic acid; and
- fusing the reconstituted particle and the liposome.
[0119] A gene transfer vector containing a protein derived from a viral envelope may also
be prepared by a method comprising the steps of:
- recombinantly expressing F protein and HN protein, in a host cell in which a protease
that processes F protein is expressed;
- isolating F protein and HN protein;
- reconstituting F protein and HN protein in the presence of a surfactant and lipids
to prepare a reconstituted particle;
- preparing a liposome filled with an intended nucleic acid; and
- fusing the reconstituted particle and the liposome.
(1.2. Preparation of a gene transfer vector encapsulating a foreign gene in a viral
envelope)
[0120] One exemplary method of preparing a gene transfer vector encapsulating a foreign
gene in a viral envelope comprises the following steps:
1) mixing a virus and a foreign gene; and
2) freezing and thawing the mixture, or mixing the mixture with a surfactant.
[0121] Alternatively, a gene transfer vector derived from viral envelope can be prepared
by a method comprising the steps of:
- inactivating a virus;
- mixing the inactivated virus with a foreign gene; and
- freezing and thawing the mixture.
[0122] In a further aspect of the present invention, there is provided a method of preparing
an inactivated virus envelope vector for gene transfer, comprising the steps of:
- inactivating a virus; and
- mixing the inactivated virus with a foreign gene in the presence of a surfactant.
(1.3. Liposome vector)
[0123] A liposome vector may use liposomes prepared from lipids commonly used in lipofection.
For example, lipids such as lipofect AMINE 2000 may be used.
(Selection of cells)
[0124] In the present invention, a variety of cells may be used as the first host cell.
The first host cells are preferably mammalian cells and more preferably cells derived
from the species from which the candidate nucleic acid is derived.
[0125] In the present invention, a variety of cells may be used as the second host cell.
Any kind of cells may be used as the second host cell. Cells suited for the second
host cell will incorporate one kind of candidate nucleic acid per cell. Examples of
such cells include, but are not limited to, bacterial and fungal cells.
[0126] In the present invention, a variety of cells may be used as the third host cell.
The third host cell is preferably the same as the first host cell, or a cell having
a similar gene expression mechanism.
[0127] The present invention has now been illustrated with its preferred embodiments. The
present invention will further be illustrated based on the Examples and referring
to the drawings attached hereto. It should be noted that the following Examples are
provided by way of illustration, and are not intended to limit the present invention.
Therefore, the scope of the present invention is not limited to any specific embodiments
recited by the examples below, and is only defined by the attached claims.
EXAMPLES
(Example 1: Preparation and use of a gene transfer vector encapsulating a foreign
gene and a component containing a protein derived from viral envelope)
(Preparation of virus)
[0128] HVJ, Z strain, was purified by differential centrifugation as previously described
(Kaneda, Cell Biology: A Laboratory Handbook, J. E. Cells (Ed.), Academic Press, Orlando,
Florida, vol. 3, pp. 50-57 (1994)). The purified HVJ was resuspended in a buffered
salt solution (BSS: 137 mM NaCl, 5.4 mM KCl, 10 mM Tris-HCl, pH7.5), and the virus
titer was determined by measuring absorbance at 540 nm. Optical density at 540 nm
corresponds to 15,000 haemocyte aggregation unit (HAU) and correlates with fusion
activity.
(Extraction of F and HN fusion protein from HVJ)
[0129] Nonidet P-40 (NP-40) and phenylmethylsulfonyl fluoride (PMSF) dissolved in ethanol
were added to 20 mL of suspension of purified HVJ (1, 750, 000 HAU), at final concentrations
of 0.5% and 2 mM, respectively. The mixture was incubated at 4°C for 30 minutes with
mixing. Then, the suspension was centrifuged at 100,000g, 4°C for 75 minutes to remove
insoluble proteins and virus genome (Uchida et al., 1979). The supernatant was dialyzed
against 5 mM phosphate buffer (pH 6.0) for three days, and the remaining NP-40 and
PMSF were removed off by exchanging the buffer every day. The dialyzed solution was
centrifuged at 100,000g, 4°C for 75 minutes, thereby removing insoluble substances.
The supernatant was applied to an ion exchange column of CM-Sepharose CL6B (Pharmacia
Fine Chemicals, Uppsala, Sweden) that was equilibrated with 10 mM phosphate buffer
(pH 5.2) containing 0.3 M sucrose and 1 mM KCl, according to the previously described
method (Yoshima et al., J. Biol. Chem., 256:5355-5361 (1981)). Flow-through fractions
and the 0.2 M NaCl eluate were collected. These fractions were subjected to sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed for protein
components. The gel was stained with Coomassie brilliant blue, and the ratio of each
protein was evaluated using computerized densitometry (NIH Image; Apple Computers,
Cupertino, CA, USA).
(Recombinant expression)
[0130] A fusion protein of HVJ may be prepared by incorporating a gene coding the fusion
protein into an expression vector and expressing the gene in an appropriate host cell.
The amino acid sequences of F protein and HN protein are known.
[0131] Expression vectors that may be used in various host cells are commercially available.
[0132] An expression vector encoding a fusion protein that may be transferred into a cell
and subsequent production of a fusion protein of the present invention is known in
the art, and can be carried out by any of the various described methods (for example,
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed, Vols. 1 to 3, Cold
Spring Harbor Laboratory Press, New York (1989) and Ausubel et al., Current Protocols
in Molecular Biology, John Wiley and Sons, Baltimore, MD (1994), each incorporated
herein by reference)). Examples of methods for transferring a recombinant expression
vector into a prokaryotic or a eukaryotic cell include electroporation methods, transformation
methods, or transfection methods.
[0133] When the recombinant Fprotein was expressed in Escherichia coli, it was expressed
in an inactive F0 form. In order to convert the inactive F0 form of protein expressed
in Escherichia coli into an active F1 form, trypsin treatment at 37°C for 30 minutes
using 0.0004-0.001% trypsin was required.
[0134] The polypeptide corresponding to activated F1 protein treated with trypsin may be
expressed in Escherichia coli using an expression vector containing a gene encoding
the amino acid sequence of a truncated, activated F1. The truncated F1 protein necessarily
includes at least 26 amino acids from phenylalanine, at position 117, to alanine,
at position 142. In the case where the truncated protein forms an inclusion body,
those skilled in the art can readily obtain an active form of the protein by refolding
the inclusion body (see Robert F. Kelley and Marjorie E. Winkler, Genetic Engineering,
(1990) vol. 12, pp.1-19 for reference).
[0135] When F protein is expressed by using cells in which HVJ can replicate (for example,
rodent tracheal epithelium; chicken embryos; f monkey kidney primary culture cells;
human fetal lung primary culture cells, kidney and amnion) as a host cell, the expressed
full length F protein is cleaved by an endogenous protease of the host cell, and as
a result, is activated. In this manner, an active form of F protein can be expressed
and isolated. Alternatively, host cells in which Tryptase clara (Kido et al., Molecular
Cells 9, 235-244 (1999)) is expressed as an endogenous enzyme (for example, rat tracheal
epithelium) or host cells in which Tryptase clara is recombinantly expressed may also
be used.
[0136] Methods of selecting and constructing these expression vectors, methods of transferring
into host cells, methods of expressing in host cells, and methods of collecting expressed
proteins are well known by those skilled in the art.
(Purification of fusion protein from HVJ)
[0137] In order to purify a fusion protein, a lysate of HVJ treated with NP-40 was clarified
by ultracentrifugation. Proteins in the supernatant were analyzed by SDS-PAGE prior
to further purification. This supernatant containedmanyproteins derived from HVJ.
Then, the supernatant was applied to an ion exchange column. Proteins of 52 kDa and
72 kDa were dominantly eluted in the flow-through fraction. These two proteins were
identified as F1 and HN, respectively, based on their mobility in an SDS-PAGE gel
(Okada, Methods in Enzymology, N. Duzgnes (Ed.), Academic Press; San Diego, vol.221,
pp.18-41 (1993)). A minor band under the 52 kDa protein was considered to be a degradation
product of the fusion proteins (Fl and HN). This is because these proteins were not
reproducibly observed in different experiments. Proteins were further eluted with
0.2 M NaCl. However, fusion proteins were not obtained efficiently. Furthermore, a
protein of 60 kDa that is speculated to be NP protein of HVJ additionally appeared.
Eventually, only the flow-through fraction was used as a source of fusion proteins
for subsequent experiments. Densitometry indicated that the F1 to HN concentration
ratio in the flow-through fraction was 2.3:1. This is consistent with the ratio of
these proteins in the viral envelope. A previous paper (Nakanishi et al., Exp. Cell
Res., 142:95-101 (1982)) reported that this ratio is required for efficient fusion
of HVJ.
(Preparation of a gene transfer vector)
[0138] A lipid mixture consisting of 3.56 mg of phosphatidyl choline and 0.44 mg of cholesterol
was dissolved in chloroform, and the resultant lipid solution was evaporated in a
rotary evaporator (Uchida et al. , J. Cell. Biol. 80:10-20 (1979)). The dry lipid
mixture was completely dissolved in 2.0 mL of protein solution (1.6mg) from the above
flow-through fraction containing 0.85% NP-40 using a Vortex mixer.
[0139] This solution was then dialyzed against 10 mM phosphate buffer (pH 7.2) containing
0.3 M sucrose and 1 mM KCl to thereby remove NP40. The dialysis was continued for
6 days, and the buffer was replaced every day. The dialyzed solution was applied to
agarose beads (Bio-Gel A-50m) (Bio-Rad Laboratories, Hercules, CA, USA) and equilibrated
with 10 mM phosphate buffer (pH5.2) containing 0.3 M sucrose and 1 mM KCl. Fractions
having an optical density of more than 1.5 at 540 nm were collected as reconstituted
fusion particles. The gene transfer vector was prepared by fusing the reconstituted
fusion particles with a liposome filled with nucleic acid prepared from 1. 0 mg of
lipids as described below.
(Expression of luciferase gene in HEK293 strain-derived transfected cells)
[0140] In order to confirm the gene transferring activity of the gene transfer vector prepared
in the aforementioned manner, HEK293 cells and a luciferase gene were used in the
following manner.
[0141] pCMV-luciferase (7.4 kb) was constructed by cloning a luciferase gene from pGEM-luc
(Promega Corp., Madison, WI, USA) into pcDNA3 (5.4 kb) (Invitrogen, San Diego, CA,
USA) at Hind III and Bam HI sites. A gene transfer vector containing about 40 µg of
pCMV-luciferase was constructed in the manner described above, and 1/10 amount (100
µL) of the gene transfer vector (about 1.5x10
11 particles/mL, DNA concentration about 40 µg/mL) was incubated with 2x10
5 cells derived fromhuman 293 cell line (human embryonic kidney: HEK). Using HVJ liposomes,
the same amount of luciferase DNA was transferred into 2x10
5 HEK293 cells. Twenty four hours after transduction, the cells were collected, and
assayed for luciferase activity in the described manner (Saeki et al., Hum. Gene Ther.,
8: 1965-1972 (1997)).
(Example 2: Preparation of HVJ envelope vector by freezing and thawing and uses thereof)
(Preparation and use of a gene transfer vector)
(1: Preparation of HVJ envelope vector by freezing and thawing)
[0142] A recombinant HVJ virus containing a luciferase gene as a foreign gene, was subjected
to various cycles of freezing and thawing before being transferred into a cultured
cell.
[0143] Five hundred µL of TE, 750 µg of luciferase expression vector pcOriPLuc (Saeki and
Kaneda et al., Human Gene Therapy, 11, 471-479 (2000)) and various concentrations
of HVJ virus were mixed. HVJ virus was prepared in concentrations of 10, 25, 50 and
100 HAU/µL. The resultant solution was divided into 12 aliquots, and each aliquot
was subjected to up to 30 cycles of freezing and thawing, each cycle consisting of
freezing by storing at 4°C, frozen with dry ice and thereafter thawed; this was repeated
up to 30 times. The resultant solution, having been subjected to a predetermined number
of freezing and thawing cycles, was added to the medium of BHK-21 cells (4×10
4 cells/dish, 0.5 mL DMEM, 10%FCS per 24-well dish), allowed to react at 37°C under
5% CO
2 for 20 minutes, the cells were washed with PBS, and then 0.5 mL of culture medium
was freshly added and the cells cultured for 24 hours.
[0144] After removing the medium, 500 µL of 1×Cell Culture Lysis Reagent (Promega) was added
to the cells to lyse the cells, and the resulting cell suspension was centrifuged
in a micro tube. Twenty µL of the obtained supernatant was measured for luciferase
activity using the Promega Luciferase Assay System and Lumat LB9501 Luminophotometer.
The measurement was conducted three times for each solution, and an average value
was determined.
[0145] As a result, it was observed that luciferase activity increased with the number of
cycles of freezing and thawing of the recombinant HVJ virus. Upon 20 cycles of freezing
and thawing, ten fold or more luciferase expression was observed as compared to 3
cycles of freezing and thawing. This result revealed that the number of cycles of
freezing and thawing of recombinant HVJ virus is preferably 5 or more, more preferably
about 15 to 20 under the conditions of the present Example.
(2: Gene transferring efficiency of HVJ envelope vector prepared by freezing and thawing)
[0146] After 30 cycles of freezing and thawing of the recombinant HVJ virus, similar to
that shown in Example 1 above, the transfer efficiency of a gene into cells was examined
under the condition wherein the number of viruses added to the host cells was constant.
[0147] For example, in the case where the X axis is 500 HAU, 50 µL of a solution having
a virus concentration of 10-50 HAU/µL, and 5 µL of a solution having a virus concentration
of 100 HAU/µL were used. The efficiency of gene expression for a solution having a
virus concentration of 100 HAU/µL was lower by about 50% compared with that for a
solution having a virus concentration of 10-50 HAU/µL. This result revealed that under
the conditions of this example, the recombinant virus concentration was preferably
in the range of 10 to 50 HAU/µL.
[0148] After 29 cycles of freezing and thawing of the recombinant HVJ virus, the 30th freezing
was conducted, the recombinant virus stored frozen for one week and then thawed before
being added to cells. The recombinant HVJ virus stored frozen for one week and the
recombinant HVJ virus subj ected to a continuous 30 cycles of freezing and thawing
showed similar levels of luciferase gene expression.
(Example 3: Preparation of an inactivated HVJ envelope vector utilizing a detergent)
(1: Growth of HVJ)
[0149] In general, HVJ cultured by inoculating a fertilized chicken egg with seed virus
may be used. However, HVJ grown in cultured cells (e.g., simian or human) or a persistent
infection system (i.e., a culture medium supplemented with a hydrolase such as trypsin
is added to cultured tissue), or HVJ grown by infecting cultured cells with cloned
virus genome to cause persistent infection are applicable.
[0150] In the present example, the growth of HVJ was performed as follows.
[0151] HVJ seed virus was cultured in a SPF (Specific Pathogen Free) fertilized egg. The
isolated and purified HVJ (Z species) was dispensed into a cryo-vial, DMSO added to
10%, and stored in liquid nitrogen.
[0152] Chicken eggs were obtained immediately after fertilization, and placed in an incubator
(SHOWA-FURANKI P-03 type; capable of accommodating about 300 chicken eggs), and incubated
for 10 to 14 days at 36.5°C and 40% or more humidity. In a darkroom, the viability
of the embryo as well as the air cell and the chorioallantoic membrane was confirmed
using an egg tester (specifically, an egg-tester in which light from a light bulb
is projected through a window having a diameter of about 1.5 cm). A virus-inj ection
site was marked in pencil about 5 mm above the chorioallantoic membrane (the position
was selected so as to avoid any thick blood vessels). The seed virus (which was removed
from liquid nitrogen) was diluted 500-fold with a polypeptone solution (1% polypeptone,
0.2% NaCl, adjusted to pH 7.2 with 1 M NaOH, then autoclave-sterilized and stored
at 4°C), and left at 4°C. The egg was disinfected with Isodine™ and alcohol. A small
hole was made in the virus-injected site with a pick. Using a 1 ml syringe and a 26
gauge needle, 0.1 ml of the diluted seed virus was injected into the chorioallantoic
cavity. Molten paraffin (melting point: 50 to 52°C) was placed onto the hole using
a Pasteur pipette in order to seal the hole. The egg was placed in an incubator and
incubated for three days at 36. 5°C and 40% or more humidity. The inoculated egg was
then left overnight at 4°C. The following day, the air cell portion of the egg was
broken with forceps, and a 10 ml syringe with an 18 gauge needle was placed in the
chorioallantois so as to aspirate the chorioallantoic fluid, which was collected in
a sterilized bottle and stored at 4°C.
(2: Purification of HVJ)
[0153] HVJ may be purified by purification methods utilizing centrifugation, purification
methods utilizing a column, or any other purification methods known in the art.
(2.1: Centrifugation-based purification method)
[0154] Briefly, a suspension of cultured viruses was collected, and the medium centrifuged
at low speed to remove tissue or cell debris in the culture medium and the chorioallantoic
fluid. The supernatant thereof was purified by high-speed centrifugation (27,500×g,
30 minutes) and ultracentrifugation (62,800×g,90 minutes) on a sucrose density gradient
(30 to 60%w/v). Care should be taken to treat the virus as gently as possible during
purification, and to store the virus at 4°C.
[0155] Specifically, in the present example, HVJ was purified by the following method.
[0156] About 100 ml of HVJ-containing chorioallantoic fluid (the chorioallantoic fluid from
chicken eggs containing HVJ, which was collected and stored at 4°C) was placed in
two 50 ml centrifuge tubes with a wide-mouth Komagome type pipette (see Saeki, Y.,
and Kaneda, Y: Protein modified liposomes (HVJ-liposomes) for the delivery of genes,
oligonucleotides and proteins. Cell Biology; A laboratory handbook (2nd edition) ed.
by J.E. Celis (Academic Press Inc., San Diego) vol. 4, 127 to 135,1998), centrifuged
in a low-speed centrifuge at 3000 rpm and at 4°C for 10 minutes (without braking)
to remove the tissue debris from the egg.
[0157] After centrifugation, the supernatant was dispensed into four 35 ml centrifuge tubes
(designed for high-speed centrifugation), and centrifuged for 30 minutesinafixed-angle
rotor at 27,000g, (with acceleration and braking). The supernatant was removed, BSS
(10 mM Tris-HCl (pH 7.5), 137 mM NaCl, 5.4 mM KCl; autoclaved and stored at 4°C) (BSS
is interchangeable with PBS) was added to the pellet in an amount of about 5 ml per
tube, and allowed to stand at 4°C overnight. The following morning, the pellets were
resuspended by gentle pipetting with a wide-mouth Komagome type pipette and collected
in one tube, and then similarly centrifuged for 30 minutes in a fixed-angle rotor
at 27,000g. The supernatant was removed, and about 10 ml of BSS was added to the pellet
and allowed to stand at 4°C overnight. The following morning the pellets were resuspended
by gentle pipetting with a wide-mouth Komagome type pipette and then centrifuged for
10 minutes in a low-speed centrifuge at 3000 rpm at 4°C (without braking), thereby
removing tissue debris and agglutinated virus which had not been completely removed.
The supernatant was placed in a fresh sterilized tube, and stored at 4°C as the purified
virus stock.
[0158] To 0.1 ml of this virus solution, 0.9 ml of BSS was added, and the absorption at
540 nm was measured with a spectrophotometer. The virus titer was converted into an
erythrocyte agglutination activity (HAU). An absorption value of 1 at 540 nm approximately
corresponded to 15,000 HAU. It is considered that HAU is substantially proportional
to fusion activity. Alternatively, erythrocyte agglutination activity may be measured
by using a solution containing (0.5%) chicken erythrocytes (see DOUBUTSU SAIBO RIYO
JITSUYOKA MANUAL (or "Practice Manual for Using Animal Cells"), REALIZE INC. (ed.
by Uchida, Oishi, Furusawa) pp. 259 to 268, 1984).
[0159] Furthermore, purification of HVJ using a sucrose density gradient may be performed
as necessary. Specifically, a virus suspension is placed in a centrifuge tube in which
60% and 30% sucrose solutions (autoclave-sterilized) are layered, and the density
gradient centrifuged for 120 minutes at 62,800×g. After centrifugation, the virus
is visible as a band at the interface of the 60% sucrose solution layer, and is recovered.
The recovered virus suspension is dialyzed overnight at 4°C against an external solution
of BSS or PBS, thereby removing the sucrose. In the case where the virus suspension
is not to be immediately used, glycerol (autoclave-sterilized) and a 0.5 M EDTA solution
(autoclave-sterilized) are added to the virus suspension so as to attain final concentrations
of 10% and 2 to 10 mM, respectively, the suspension is then gently frozen at -80°C,
and finally stored in liquid nitrogen (the frozen storage can be performed with 10
mM DMSO, instead of glycerol and a 0.5 M EDTA solution).
(2.2: Purification method utilizing columns and ultrafiltration)
[0160] Instead of purification by centrifugation, purification of HVJ utilizing columns
is also applicable to the present invention.
[0161] Briefly, concentration (about 10 times) via ultrafiltration utilizing a filter having
a molecular weight cut-off (MWCO) of 50,000 and elution via ion exchange chromatography
(0.3 M to 1 M NaCl) were performed to achieve purification.
[0162] Specifically, in the present example, the following method was used to purify HVJ.
[0163] After the chorioallantoic fluid was collected, the chorioallantoic fluid was filtrated
through a membrane filter (80 µm to 10 µm). To the choricallantoic fluid, 0.006 to
0.008% BPL (final concentration) was added (4°C, 1 hour), so as to inactivate the
HVJ. The chorioallantoic fluid was incubated for 2 hours at 37°C, thereby inactivating
the BPL.
[0164] About 10 times concentration was achieved using a tangentialflow ultrafiltration
method using a 500KMWCO membrane (A/G Technology, Needham, Massachusetts). As a buffer,
50 mM NaCl, 1 mM MgCl
2, 2% mannitol, and 20 mM Tris (pH 7.5) were used. An HAU assay indicated an HVJ yield
of approximately 100%. Thus, excellent results were obtained.
[0165] HVJ was purified by column chromatography (buffer: 20 mM Tris HCl (pH 7.5), 0.2 to
1 M NaCl) using a Q Sepharose FF column (Amersham Pharmacia Biotech KK, Tokyo). The
yield was 40 to 50%, and the purity was 99% or more.
[0166] An HVJ fraction was concentrated by tangential flow ultrafiltration using a 500KMWCO
membrane(A/G Technology).
(3: Inactivation of HVJ)
[0167] In the case where it was necessary to inactivate HVJ, this was performed by UV light
irradiation or treatment with an alkylating agent, as described below.
(3.1: UV light irradiation method)
[0168] One milliliter of HVJ suspension was placed in a dish having a diameter of 30 mm,
and subjected to an irradiation at 99 or 198 mJ/cm
2. Although gamma-ray irradiation is also applicable (5 to 20 Gv), it does not provide
complete inactivation.
(3.2: Treatment with an alkylating agent)
[0169] Immediately before use, 0.01% β-propiolactone was prepared in 10 mM KH
2PO. The solution was kept at a low temperature during preparation, and the operation
was quickly performed.
[0170] β-propiolactone was added to a final concentration of 0.01% to the HVJ suspension
obtained immediately after purification, and the mixture was then incubated on ice
for 60 minutes. Thereafter, the mixture was incubated at 37°C for 2 hours. The mixture
was dispensed into Eppendorf tubes in 10,000 HAU aliquots, and centrifuged for 15
minutes at 15,000 rpm. The precipitate was stored at -20°C. Instead of using the aforementioned
inactivation method, without storing the precipitate at -20°C, DNA may be incorporated
into a vector by detergent treatment alone when constructing a vector.
(4: Construction of an HVJ envelope vector)
[0171] To the HVJ which had been stored, 92 µl of a solution containing 200 to 800 µg of
exogenous DNA was added, and well mixed by pipetting. This solution can be stored
at -20°C for at least 3 months. By adding protamine sulfate to the DNA before mixing
with HVJ, the expression efficiency was enhanced twofold or more.
[0172] This mixture was placed on ice for 1 minute, and 8 µl of (10%) octylglucoside was
added. The tube was shaken on ice for 15 seconds, and allowed to stand on ice for
45 seconds. The treatment time with the detergent is preferably 1 to 5 minutes. Instead
of octylglucoside, detergents such as Triton-X100(t-octylphenoxypolyethoxyethanol),
CHAPS(3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate), or NP-40 (nonylphenoxy
polyethoxy ethanol) may also be used. The final concentrations of Triton-X100, NP-40,
and CHAPS are preferably 0.24-0.80% (v/v), 0.04-0.12% (v/v) and 1.2-2.0% (v/v), respectively.
[0173] One milliliter of cold BSS was added, and the solution was immediately centrifuged
for 15 minutes at 15,000 rpm. To the resultant precipitate, 300 µl of PBS or saline,
etc., was added, and the precipitate suspended by vortexing or pipetting. The suspension
may be directly used for gene transfer or may be used for gene transfer after storage
at -20°C. After being stored for at least 2 months, this HVJ envelope vector maintained
the same level of gene transfer efficiency.
(Gene transfer method)
[0174] An amount of vector equivalent to 1, 000 HAU (30 µl) was placed into an Eppendorf
tube, and 5 µl of protamine sulfate (1 mg/ml) was added. The medium was removed from
BHK-21 cells (which were sown in 6 well dishes at a density of 200,000 cells per well
the previous day) and 0.5 ml of medium (10%FCS-DMEM) was added to per well. To each
well, a mixture of the aforementioned vector (equivalent to 1,000 HAU) and protamine
sulfate was added, and the plate was shaken back and forth and from right to left,
whereby the vector and cells were well mixed. The mixture was left in a 5%CO
2 incubator for 10 minutes at 37°C.
[0175] The medium was replaced, and the cells were left overnight (16 hrs to 24 hrs) at
37°C in a 5% CO
2 incubator, after which the gene expression was examined. To measure luciferase activity
(pcLuci: a luciferase gene having a CMV promoter), the cells were lysed with 0.5 ml
of Cell Lysis Buffer (Promega), and the activity in 20 µl of the solution was measured
using a luciferase assay kit (Promega). To measure green fluorescent protein activity
(pCMV-GFPE; Promega), the cells were observed under a fluorescent microscope in their
intact form, and 5 to 8 fields were observed at a magnification of 400, and the ratio
of cells which generated fluorescence was calculated.
(Example 4: Preparation and use of a liposome vector)
(Preparation of liposome vector)
[0176] A liposome vector of the present invention is prepared as follows: Twenty to twenty
four µg of cDNA library-derived cDNA and 24-72 µL of lipofect AMINE 2000 reagent (In
vitrogen life technologies (Carlsbad, California 92008) are respectively diluted in
1.2 mL of serum free medium, and rapidly mixed. The mixture is incubated at room temperature
for 20 minutes, to form a complex of liposomes and nucleic acid.
(Transfection by liposome vector)
[0177] Host cells are added to each well of 96-well plate together with an appropriate medium,
and cultured. When transfection is conducted in the presence of serum, 12.5 µL of
the transfection complex is directly added to each well of the 96-well plate and mixed.
When transfection is conducted in absence of serum, the medium containing serum is
removed and replaced by a serum free medium before adding the transfection complex.
After incubation in a CO
2 incubator for 4 to 12 hours, the medium is replaced. After a predetermined period
of culture, an assay is conducted.
(Example 5: Isolation and analysis of a gene encoding vascular endothelial growth
factor from a human heart cDNA library.)
[0178] Using the present invention, it is possible to isolate a gene of interest having
an intended functional property. One embodiment of the isolation method is schematically
shown in Fig. 1.
[0179] Actually, using the gene transfer vector prepared in Example 3 of this specification,
a gene encoding vascular endothelial growth factor was isolated. For isolating the
gene exemplarily shown in this example, not only the gene transfer vector prepared
in Example 3 of this specification, but also any "viral envelope vector" and "liposome
vector" may be used.
[0180] Human heart cDNA library (GIBCO BRL; plasmid prepared by ligating human heart-derived
cDNA to plasmid pSPORT having a CMV promoter) was transferred into E. coli DH12S,
and the plasmid was prepared from the E. coli. Two hundred µg of plasmid was encapsulated
in 10000 HAU of HVJ-E gene transfer vector (gene transfer vector prepared in Example
3 of the invention, 3 x 10
9 particles). About 5000 human aortal endothelial cells (HAEC) (Sanko Junyaku) were
added to each well of a 96-well micro titer plate together with growth medium and
cultured overnight. The cultured cells were used as host cells. To each well containing
host cells, 1/100 amount of the above HVJ-E was added. The wells were kept at 37°C
for 30 minutes, and then the medium was replaced.
[0181] The medium used was a low nutrient condition medium having a serum concentration
of 1%. Under these conditions, culture was conducted for one week. Under these conditions,
growth of HAEC was not observed.
[0182] After two weeks, a cell growth assay was conducted. Using Cell Titer·96 (Promega)
as a regent, cell growth was evaluated based on the color change that is indicative
of the redox state of mitochondria. The result is shown in Fig. 2.
[0183] In Fig. 2, the wells having the deepest color are wells where cell growth occurred
most actively. The entire micro titer plate was read with a plate reader, and cell
growth was graphically shown with a computer as shown in Fig. 3. DNA was extracted
from cells in the two wells exhibiting the greatest growth according to the graph,
using a DNeasy Tissue Kit available from Qiagen. Since the prepared nucleic acid includes
plasmid DNA, it was transferred into competent E. coli (DH5α; TAKARA) by heat shock.
[0184] This E. coli was inoculated on an ampicillin-containing solid media, and allowed
to form colonies. From DNA prepared from a single well, about 20-200 colonies were
obtained. Plasmid DNA (pDNA) was extracted from each colony, and the presence of a
gene fragment in the plasmid was confirmed by restriction enzyme analysis. About 60-70%
of plasmids in the prepared plasmids were plasmids had an insert (the white arrow
in Fig. 4 shows an insert fragment).
[0185] Next, plasmid DNA was purified using Endo Free Plasmid Maxi Kit available from Qiagen,
and the purified plasmid was encapsulated in HVJ-E and transferred into HAEC cells
again, and a cell growth test similar to that described above was conducted. In this
cell growth test, the plasmid exhibiting significantly high cell growth is a candidate
plasmid that is expected to include a nucleic acid encoding vascular endothelial growth
factor. In the present example, two clones (p3743, p77421) exhibited high HAEC growth
activity with high reproducibility. Results from one of these clones is shown in Fig.
5.
[0186] The gene products isolated in the present experiment had higher growth activity than
VEGF or HGF with respect to human aortal endothelial cells HAEC. However, they exhibited
similar activity with VEGF or HGF with respect to human vascular smooth muscle cells.
(Discussion of angiogenetic activity)
[0187] The foregoing two genes exhibiting high HAEC growth activity (p3743, p77421) were
evaluated for angiogenetic ability using an Angiogenesis Kit, KZ-1000 (KURABO INDUSTRIES
LTD.) in accordance with the following procedure. As controls, blank, vascular endothelial
growth factor-A protein (VEGF-A), pVEGF plasmid (a plasmid containing a gene encoding
vascular endothelial growth factor) and pSPORT1 (a plasmid not containing a gene encoding
vascular endothelial growth factor) were used. Each obtained image was quantified
using an angiogenesis quantification software (KSW-5000U, KURABO INDUSTRIES LTD.)
in accordance with the following procedure.
[0188] On a 24-well plate, an angiogenesis KIT KZ-1000 (KURABO INDUSTRIES LTD.) co-culture
human umbilical vein endothelial cell and human adult skin-derived fibroblast was
used. To a medium specified for angiogenesis (KZ-2400, KURABO INDUSTRIES LTD.) 10
ng/mL of VEGF-E (NZ-7) was added, and anti human VEGF-A neutralizing antibody was
added in concentration of 0, 250, 500, 1000 ng/mL and the medium used to culture the
above cells.
[0189] Anti-VEGF, Human, Mouse-Mono (26503.111) (R&D, Catalog No. MAB293) was used as the
anti human VEGF-A neutralizing antibody. Culture was conducted at 37°C in a 5% CO
2 incubator. After 4, 7 and 9 days of culture, the medium was replaced with fresh media
supplemented with the same additives. After 11 days of culture, the medium was removed,
and staining was conducted using a lumen staining kit (for staining CD31 antibody:
KURABO INDUSTRIES LTD. KZ-1225) in accordance with the following procedure.
[0190] CD31 (PECAM-1)-staining primary antibody (mouse anti-human CD31 antibody) was 4,000-fold
diluted in blocking solution (Dulbecco phosphate buffer (PBS (-) containing 1% BSA).
To each well, 0. 5 mL of this primary antibody solution was added, and incubated for
60 minutes at 37°C. After incubation, each well was washed a total of three times
with 1 mL of blocking solution.
[0191] Then, 0.5 mL of a secondary antibody solution (goat anti-mouse IgG alkaline phosphatase
complex) that was 500-fold diluted with a blocking solution was added to each well,
incubated for 60 minutes at 37°C, and then washed three times with 1 mL of distilled
water. During this, two tablets of BCIP/NBT were dissolved in 20 mL of distilled water,
and filtered through a filter having a pore size of 0.2 µm, to prepare a substrate
solution. Then 0.5 mL of the prepared BCIP/NBT solution was added to each well, and
incubated at 37°C until the lumen turned deep violet (usually 5 to 10 minutes). After
completion of incubation, each well was washed three times with 1 mL of distilled
water, and the washing solution removed by aspiration. Then each well was left to
stand in order to air dry. After drying, each well was observed under a microscope.
[0192] Each well was observed under x40 magnification, and photographed.
[0193] A picture in which a scale of 1 mm magnified 40-folds was taken (Fig. 6), and based
on this scale, the area of the lumen (left in Fig. 7), the length of the lumen (right
in Fig. 7), the joints of the lumen (left in Fig. 8) and the path of the lumen (right
in Fig. 8) formed in each visual field were measured. The number of branch points
of the lumen is denoted by "joint" and the number of lumens coming from the branch
point is denoted by "path".
[0194] The observation result shown in Fig. 6 and the date of the lumen formed shown in
Figs. 7 and 8 demonstrated that clone p77421 has a similar degree of angiogenetic
activity to VEGF, and the clone p3743 has better angiogenetic activity than VEGF.
(c-fos luciferase assay)
[0195] The influence that the product of the genes isolated in the foregoing experiment
exerts on the activity of promoter of c-fos gene was examined by reporter assay using
a reporter plasmid incorporating a c-fos gene promoter upstream of a luciferase gene.
[0196] Specifically, the assay was conducted in the following manner.
[0197] Endothelial cells were seeded in a 6-well plate, and transfected with a c-fos-luciferase
reporter gene (p2FTL) using lipofect AMINE PLUS (GIBCO-BRL). This fos-luciferase reporter
gene consists of 2 copies of the c-fos 5'-regulatory enhancer element (-357 to -276),
the thymidine kinase gene promoter of herpes simplex virus (-200 to +70), and a luciferase
gene. It was co-transfected with p3743 plasmid as necessary. Twenty four hours after
transfection, transfected cells were incubated in a serum free medium for 24 hours.
As necessary, cells in rest state were treated with 100 ng/ml of HGF (hepatocyte growth
factor) or with GFP (green fluorescent protein) for four hours. After washing with
PBS and adding with 500 µL of cell lysis buffer, the cells were kept at room temperature
for 15 minutes, and thereby lysed. 10 µL of cell extract obtained by lysing cells
was mixed with 100 µL of luciferase assay reagent, and light emission was measured
for 30 seconds using a luminometor in units of RLU. The significant increase in luciferase
activity demonstrated that p3743 increases c-fos gene promoter activity (Fig. 9).
[0198] The above result demonstrates that a gene having an intended functional property
may be readily and conveniently isolated by using the present invention.
(Example 6: Isolation method of mutant nucleic acid having an intended functional
property)
[0199] With the present invention, it is possible to isolate a mutant gene having an intended
functional property.
[0200] Using a gene transfer vector prepared in Example 3 of this specification, a gene
encoding vascular endothelial growth factor is isolated. For isolating the gene exemplarily
shown in this example, not only the gene transfer vector prepared in Example 3 of
this specification, but also any "viral envelope vector" and "liposome vector" may
be used.
[0201] As a starting material, a nucleic acid comprising a specified gene is selected. A
plasmid in which the selected nucleic acid is operably linked to a sequence that functions
as a promoter in a first host cell is constructed. The plasmid is transferred into
a first host cell according to Example 3.
[0202] The first host cell into which the nucleic acid is transferred is subjected to mutagenesis,
and about 5000 cells are added on each well of 96-well micro titer plate together
with a medium, followed by overnight cultivation. After cultivation, mutated host
cells in each well are screened for an intended function. Cells having an intended
function are isolated, and nucleic acid is extracted from the cells in the well exhibiting
the most desired property using a DNeasy Tissue Kit available from Qiagen. Since the
prepared nucleic acid includes plasmid DNA, it is transferred into competent E. coli
(DH5α; TAKARA) by heat shock transformation.
[0203] This E. coli is inoculated onto ampicillin-containing solid media, and allowed to
form colonies. From DNA prepared from a single well, about 20-200 colonies can be
obtained. Plasmid DNA is extracted from each colony, and the presence of a gene fragment
in the plasmid is confirmed by restriction enzyme digestion.
[0204] Then, using an Endo Free Plasmid Maxi Kit available from Qiagen, plasmid DNA is purified,
and the purified plasmid is encapsulated in HVJ-E and transferred into HAEC cells
again, and a similar functional property is examined. In this cell growth test, the
plasmid exhibiting a preferred functional property is recognized as a plasmid containing
a mutant nucleic acid having an intended functional property.
[0205] Although the present invention has been described in its preferred embodiments, it
should be noted that the present invention is not limited to these embodiments. Thus,
it can be understood that the scope of the present invention is defined only by the
claims attached hereto. It can be understood that those skilled in the art can practice
an equivalent scope based on the description of the present invention and the common
general knowledge in the art in view of the description of the specific preferred
embodiments. It is appreciated that patents, patent applications and documents cited
in this specification are incorporated herein by reference as if the content thereof
is specifically described in the present specification.
Industrial Applicability
[0206] A novel method and a kit for isolating a nucleic acid having an intended functional
property are provided. As a result, it is possible to isolate a nucleic acid having
an intended functional property more rapidly and conveniently than the conventional
method. The present invention is particularly effective for expression screening using
mammalian cells as a host cell.
[0207] In the present invention, by changing the combination of an objective cell and an
assay method, it is possible to isolate genes having a variety of different functions.
Specific examples of such genes include tumor-suppressor genes; osteogenesis enhancer
genes; apoptosis trigger genes; cytokine secretion genes; nerve cell dendrite inducer
genes; arteriosclerosis suppressor genes; diabetes suppressor genes; autoimmune diseases
suppressor genes; Alzheimer's disease suppressor genes; Parkinson's disease suppressor
genes and nerve cell protecting genes and the like.